Revision as of 02:59, 22 October 2019

Progress Indicator Animation

Summary
All together, we came into contact with 50+ experts and stakeholders out of more than 18 different countries from all continents. Their contributions shaped our project into what it is now and the various backgrounds of our experts and their sometimes contradictory opinions elevated our project to a real-world application that aroused interests in farmers, mycologists and experts from the industry alike. Furthermore, we were able to improve biosafety and biosecurity aspects of our project according to the legal situation, guidelines and suggestions for improvements through experts. By doing so, we made sure our Troygenics do not pose any harm for the environment or the consumer.

	General Experts
	Phage Assembly
	Endocytosis
	CeDIS
	Lab Application

Forward farm

On the 26th of September 2019, we visited the Bayer Forward Farm in Rommerskirchen

We visited the Bayer Forward Farm in Rommerskirchen, the “Damianshof” accompanied by Bernd Olligs (farmer and owner of the farm), Dr Patrick Beuters (Product Development Manager for fungicides in cereals, sugar beet and potatoes) and Karl Eschenbacher (Head of Bayer Forward Farming).
The farm in Rommerskirchen is one of two locations in Germany where Bayer established a Forward Farm: An experimental testing area for novel approaches to solve problems in the field of agriculture. Thereby, the farm’s purpose is also to educate the public about farming and emerging technologies. The engagement with the public and the promotion of face to face interaction is one of the foundations the project is based on.
During our visit, we received extensive first-hand insights into agriculture from different points of views. The different experts who accompanied us each represented a different important aspect of modern agriculture.

Dr Patrick Beuters

He is in the department of Market Development Manager for fungicides in cereals, sugar beet and potatoes.

Dr Patrick Beuters gave us insights into the importance of fungicides for agriculture. As a consultant for fungicides for cereals, sugar beet and potatoes he has an extensive overview about all their important products and modes of actions. His explanations regarding the history and recent change in usage of fungicides as well as the latest interests in research completed the depictions of Karl Eschenbacher and Bernd Olligs very well. At the same time, he stressed the need to investigate alternatives to meet future challenges. Furthermore, he showed us the extend of the recent interests of the industry for new alternatives, e.g. biologics. According to Dr Beuters, it is important to work closely with farmers to ensure proper use of fungicides and effective food safety.

Karl Eschenbacher

He is Head of Bayer Forward Farming.

Karl Eschenbacher is the Supervisor Head of Bayer Forward Farming in Germany. He stressed the importance of a close cooperation between industry, farmer and consumer. A lack of understanding can easily lead to problems and misconceptions according to him. He explained how valuable open communication about agricultural measures are to educate people about the necessity of certain methods and applications, e.g. the use of pesticides. To reduce prejudices towards agriculture, engagement and education with the public are among the greatest tools to achieve a well-understood, fair-regulated, sustainable and safe food production. Considering we live in a time, where technological advancements are achieved quickly and should be easily accessible and understood by the public, engagement with everyday people is often sparse. An open conversation about such topics and new approaches to communicate the subject in a target group specific manner can really make a difference. To sum up, Karl Eschenbacher clarified how important well-informed consumers are to achieve an integrated agriculture.

Bernd Olligs

Is the farmer that is taking care of the farm Damianshof, where the forward farm is located

Bernd Olligs is the owner of the farm. He gave us great first-hand insight into the work as a farmer. He showed us how full of meaningful decisions his work is and underlined his statements with stories and anecdotes from his work. His depictions of the life as a farmer made us realise how underestimated and unappreciated the work of farmers is in modern society and how complex the issues can be that they are confronted by. They have to assure a maximum yield while minimizing the costs and the impact on the environment while planning years far ahead. This is a task unmatched in modern society. During our visit, Bernd Olligs also described the importance of improving the personal contact between farmers and consumers to create a broader understanding of the reasons that drive their decisions and our dependency on their experience.

First Contact: 05.09
We approached Professor James Brown as an expert for fungal diseases of crops and other plants to receive an evaluation of our project as well as our concept for a potential application.
Prof. Brown gave us advice on various points of our project, which he depicted as an interesting approach that should quite certainly be a completely novel approach.
First of all, he portrayed that fungicides, although they can be unspecific and have off-target effects, are generally quite specific in their mode of action. Moreover, off-target effects are rather rare and usually weak or hard to detect. Besides this, we would have to put thought into how our system could have similar side effects, he explained. Also, he pointed out that specificity towards a certain pathogen, as well as a broader specificity can both be advantageous. For our system we would have to argue why it would be beneficial to have such a specific mode of action and off-target effects would be important enough to be worth avoiding.
Also, he pointed out, that the process of safety testing for fungicides is a rigorous, expensive and time-consuming one. During this process, the fungicides are extensively tested for potential negative impacts for the environment and for humans. This procedure takes nine to ten years after the discovery of the fungicide molecule. These tests are also an important reason, why fungicides generally have very few off-target effects if they are allowed to be commercially used. The patent life of fungicides lasts 17 years. Hence, the companies have only seven to eight years to significantly profit from the sales of a new fungicide while it is still in patent. This period can only be extended by patenting the manufacturing process or keeping important features of the product secret. The process for testing medical pharmaceuticals is quite analogous, although the period of safety testing and is two years shorter, he added. If our Troygenics would be aimed for a commercial use we should put more thought into product marketing and a strategy for IP protection to make sure to make a profit on the system, he suggested.
In his view, the short patent life of fungicides is also contributing to the problem of resistance because to make a quick return on their investment, companies have to sell as much of their product as fast as possible. The same effect applies to medical antibiotics, which results in them not being attractive investments for pharma companies anymore. For a commercial application a way to extend the patent life of the Troygenics would thereby be beneficial to maximise the potential profits.
If we knew of any possibility that fungi could evolve resistance to our Troygenics this would also have to influence our marketing strategy, Prof. Brown mentioned.
Regarding mathematic models he named us two further contacts to come in touch with.
One thing Prof. Brown especially pointed out, was that non-specific effects of fungicides are actually often advantageous and thereby desired by farmers because this substitutes multiple treatments with different pesticides. This way a crop can be sprayed just once and still be well protected from multiple diseases. Prof. Brown proposed, that the most important limitation for disease management on the fields is the number of opportunities over the course of a year, when the ground is dry enough for the usage of a tractor but not to dry, so plants are not getting damaged because of the drought. Only then, it is possible to spray against crop diseases of local importance. The conditions to do this are really fairly specific. There should be no rain at the moment and no rain expected for the next 12 to 14 hours at wind speed forces of 1-3. For crops in greenhouses, there is more flexibility, but the number of sprays is aimed to be minimized because of two reasons. Firstly, the cost of spraying, the staff and the fuel should be minimized. And secondly, crops with fewer treatments of reagents like fungicides are more attractive for supermarkets. Prof. Brown also gave a distinct for this development: During the 34 years of his career the average number of sprays applied per year to blackcurrants, an important crop where he lives, dropped from 37 to one.
Moreover, there has been concerns about negative, non-specific effects of fungicides on soil microbiota but far to less research on the subject. Prof. Brown portrayed, that he would consider a larger effect of the fungicides very unlikely. The amount of fungicides sprayed per unit area is very low. Furthermore, fungicides are usually applied when the crop canopy is well-formed and the great majority of the spray lands on the plant.
While there are some advantageous in having a very specific targeted method to fight certain crop pathogens, there are also define disadvantageous. It is important to know when such a system would be beneficial and when it would not be, Prof. Brown concluded.
The biggest current concern, regarding the off-target effects of fungicides is that triazole fungicides, which are targeting ERG11 (CYP51) in the ergosterol synthesis pathway in fungi, can have off-target effects on at least one enzyme involved in the hormone synthesis in mammals. These effects are still very small though and far below the level detectable by epidemiological analysis. The EU legislation still requires strictly no off-target effects on the mammalian reproductive system, by whatever method of testing.
This behaviour has made the companies, that develop new fungicides, very nervous, since the increasing threat of losing huge amounts of invested money because of unknown off-target effects. Thereby, a system like ours would have to prove that it does not have any off-target biological and biochemical processes, especially regarding the mammalian reproductive system in any way.
Since the interest in fungicide analogues is quite high, a range of methods have been suggested. But these are most often not as effective as the fungicides and for example require multiple applications on the field. Also transporting and application cost would increase.
Further very important characteristics of our method would be its efficacy and therefore the likely cost of the application. Additionally, a reinfection of the plant after the application should be expected. Also, it would be important to know how long our Troygenics would take to degrade and thereby how many applications would be necessary to effectively protect the crop.

First contact 09.09 Phone call 10.09 Video conference 13.09 Paul Zabel works at a Research Associate for the German Aerospace Center (DLR) in the Space Segment Systems Analysis department and has been part of a unique pilot experiment, the EDEN ISS project. The objective of this project, founded by the European Commission under the Horizon 2020 research programme, was to test a special laboratory suited for plant breeding and food production on future space missions. To test this laboratory under extreme conditions Paul Zabel spent one year in Antarctica near the AWI's Neumayer Station III to cultivate vegetables and herbs.
We approached him to receive an evaluation on the impact of fungi under these special conditions, as well as the precautions that have to be taken to prevent plant damaging fungi.
After we called Paul Zabel, we organised a video conference to discuss the role of fungi during his expedition and some aspects of our project.
There have been two shifts on the EDEN ISS since the start of the project, one in 2018 and one in 2019. A season lasts from February to November. Paul Zabel spent the very first season at the South Pole for this project.
In both years, there have been problems with fungi on the station. During the first months of its use the fungi display a faster growth and can form biofilms in the nutrient solutions of the plants. Under these conditions, fungi can start to grow on moist parts of pipes and uneven surfaces. Over time the growth of the fungi reaches a point of self-regulation. During both years, chlorine cleaner was used to counteract this problem with only moderate success. In the next years, the usage of such methods is planned to be reduced to a minimum. Samples from plant surfaces and the water have been taken and are being analyzed by the Astrobiology Group of the Institute of Aerospace Medicine in Cologne. Still the general burden of fungi on vegetables produced in the Antarctic has shown to be 1000 – 10000 times smaller than the one of store bought vegetables. Analyzed contamination samples at the arctic greenhouse rather contained spores of fungi than bacteria.
For the plants, these fungi do not pose severe threats, only the optics of some plants are affected by the fungi. But beyond that, pipes and filters can become clogged through the biofilms and can cause technical issues. Also, the plants are growing in special cubes out of mineral wool that can become overgrown by fungi. This can potentially lead to the stem of the plants to soften up. As a result, the stems of healthy plants can break and the plant dies. Still this issue only affects a small number of plants per year.
At the moment, the seeds are not being sterilized or treated in any special way, since most of the surroundings are clean surfaces, also the cubes of mineral wool are being heat-treated before use.
It is still under discussion, how extensive the sterilization process for later space missions would have to be. Since, on the International Space Station (ISS), everything is mandatory being sterilized and there are still fungal contaminations occurring in space, Zabel reports. Besides that, growing plants can not be sterilized. In the Antarctic, people are visiting the greenhouse with protective clothes, are not allowed to touch anything and have to follow certain rules like “no food” as an “intermediate solution”. This approach could be adapted for space missions later on. Cleaning and sterilization procedures are time-consuming and expensive and will be avoided if possible. In addition to this, vapors of cleaning agents would have to be filtered out in space.
The question arises, if a sterilization of a garden in space would even be possible. For example, on the ISS, four salads have been grown, sterilized with chlorine solution and water and eaten. But it is a rather minor effort to sterilize four salads. The greenhouse Pail Zabel worked in has a base area of 12,5 m2 and would be almost impossible to completely sanitize. Moreover, the water consumption of the sterilization process is quite high. To sterilize 1,5 kg of rocket (salad) around 60-70 l of water are needed, although the salads shelf life expands for about one or two weeks treated this way. So, it has to be evaluated, if the sterilization process would be worth all of this and if the approach sterilizing everything on space missions could actually be continued on this level. After all, the vegetables grown in space could still be eaten without any sterilization process. Maybe the rules on this have to be loosened a little bit, Paul Zabel concludes.
Tests have been conducted with ozone lead into the nutrient solution to lower the growth of fungi. But this has led to the binding and flocculation of nutrients like Iron or Calcium, making them inaccessible for the plants. For future mission in the Antarctic, further measures are already planned. Like tests regarding the sterilization of the seeds and the usage of hydrogen peroxide solution with silver ions added. Also, biological approaches are planned to promote a natural balance and the usage of other strains of fungi to keep the harmful ones at bay could also be a potential measure.
One thing Paul Zabel also pointed out, was that the need for the sterilization of the plants would considerably limit the selection of the plants, since the plant surface has to be accessible for the fungicide. While a salad is quite easy to sterilize, this is almost impossible for crops like carrots or potatoes because they need to be in direct contact with the nutrient solution. Research on soil-free cultivation methods of these plants is being conducted but showed to be rather challenging. Keeping the current situation of plant breeding in space in mind, there could actually be an application of our Troygenics for future space missions. Since the ultimate goal of a colony on, for example, Mars would be the total self-sufficiency of the colony, the supply on reagents to fight plant pathogens would be extremely limited. A possibility to produce targeted reagents, that only work on the fungi that are harming the plant without harming any of the beneficial fungi for the plant or the plant itself could be a huge benefit. Furthermore, our Troygenics could be modified, adapted and produced in a laboratory in space using E. coli. As a result, the colony could operate independent from supplies from earth. Since the dependency on the own harvest would be way more important in a self-sufficient space colony and the loss of plants through pathogens, fungi, or even radiation in space should be limited to an absolute minimum, our system could contribute to ensure the protection of crops under these conditions. Avoiding technical difficulties, like clogged pipes or filters would be another important issue, since repair parts are often not available.
The ability to manufacture tools or measures to solve problems, like the Troygenics, to assure the self-sufficiency of the colony in space is extremely important, Paul Zabel resumes.

First contact 25.06
Phone call 19.08
We contacted Prof. Dr. Karl-Heinz Kogel, head of the institute for phytopathology at the University of Giessen (Germany), to gather additional information on fungal crop pathogens, their impact and their ecological and economic value.
After we explained what the iGEM competition is and our project we discussed the different parts of our work.
Prof. Kogel pointed out, that our project has to be planned with a lot of foresight, since the current political and social opinion on genetic engineering would prevent such an application in the near future. With the legal situation regarding this topic in the EU he did not expect a system like our Troygenics to be applicable anytime soon.
Besides that, he still liked our approach and considered it a creative idea. Although, he also mentioned, that most comparable approaches do not try to solve this many problems at once, which makes our project susceptible to more problems in direct comparison.
He was especially interested in our approach of an induced endocytic uptake into the pathogenic fungi via specific ligands on the surface of the Troygenics.
As an additional, similar approach he proposed the idea of using only dsRNA instead of the Troygenics and gene silencing instead of CRISPR/ Cas. While this concept would elute some of the problems of our system, like the required uptake of, in comparison, larger particles into the target cell, it would also establish new ones. On one hand this would prevent GMOs to form on the fields. This would make it more realistic to implement and is closer to the current praxis. On the other hand, this would require the use of siRNAs which are distinctly more instable and way larger amounts of them would be needed to achieve the same effect. Moreover, this alternative approach would lack the shuttle our concept provides. Finally, the biological production of the Troygenics via E. coli would be a clear advantage in direct comparison.
Although, this was a great suggestion, we still considered our approach as a better solution to generate a broader range of potential applications.
Furthermore, Prof. Kogel gave us additional information about pathogenic crop fungi and their impact on agriculture. As a further potential target, that would be important to come by, he named Fusarium graminearum as an important pest of wheat and corn plants all around the world. He even mentioned some genes of certain pathways to target for our system. In this case, the pathogen usually is treated with azole-based pesticides, which deactivate proteins essential for the infection of the plant.
F. graminearum has become a substantial threat to the food security of the named crops.
Regarding Puccinia graminis, a devastating wheat pathogen, that managed to develop multiresistant strains and is threatening the food supply in parts of Africa and that is spreading continuously, he confirmed the danger of the pathogen and added, that its impact is going to increase dramatically because of the progressing climate change.
Prof. Kogel also confirmed, that problems due to fungicide resistance are already a relevant threat to our food security and supply. A far range of papers are published about this topic.
At the end, we asked Prof. Kogel about methods to distinguish between different spores of fungi and to determine their species without sequencing parts of their genome. Prof. Kogel explained, that the morphology of the spores of different fungal species are quite easy to distinguish with a closer investigation via microscopy.

First contact 04.10
Phone call 16.10
We approached Prof. Haberlah-Korr because of her extensive knowledge about plant protection and ecology and wanted to ask her about the impact fungicides have on ecosystems.
Prof. Haberlah-Korr pointed out, that the impact of the fungicide depends massively on the kind of fungicide that is used as well as its mode of action. Besides that, fungicides can have negative side effects regardless of if they are biological or chemical reagents. Biological alternatives like copper particles are widely used. These particles can accumulate in the soil but are not harmful for humans in the doses they are used in.
A very broad range of fungicides is used with very different modes of action. Some of them can have insecticidal off-target effects or can have negative impacts on soil or water systems.
Despite the public opinion, fungicides do not have a massive negative influence if they are used properly. Besides that, their usage can be justified in many cases, since our level of food production would not be feasible without them.
Many fungicides work quite specific and do not pose a threat through off-target effects. Others, like many azoles, have a broader mode of action and can even show endocrine effects in mammals. Recently, many fungicides have been revaluated because of stricter regulatory limits, but still for example endocrine effects are difficult to measure.
The Federal Office of Consumer Protection and Food Safety monitors the effects of different fungicides and their potential hazards.
Meanwhile, farmers often try to avoid fungicides by more carefully choosing their crop strains or are using resistant plants.

First Contact 05.09
We reached out to Dr. Singh as a contact at the International Maize and Wheat Improvement Center (CIMMYT), an international non-profit agricultural research and training organization that is connecting scientists and research programs worldwide to advance in crop protection for the two most important cereal grains in the world: maize and wheat.
Dr. Singh pointed out, that the International Maize and Wheat Improvement Center has mainly set its focus on keeping damages through fungal pathogens under control by using host resistances of the plants. Furthermore, fungicides are rarely used by smaller farmers in Asia or Africa. Keeping this in mind, the usage of our Troygenics would be quite complicated to realise in those African and Asian regions, since there is no infrastructure to distribute our system in an easy way. Also, our Troygenics are designed in a way, that they could be applied with the tools that are commonly used by farmers to apply fungicides, but without them being used, distributing the respective tools would be an additional problem.
Another point he criticized, was that the targeting and neutralizing of one single fungal pathogen would not avert the threat of fungal damages for the crop. Because of the high variety of fungal pathogens in a country or across countries. To effectively protect the crops a broader applicability would probably be necessary, he concludes. Beyond that, in his opinion this method would have to be implemented into the host plant but like our system, this would be genetic engineering and Dr. Singh did not expect such a measure, based on transgenic organisms, to be applicable in the real world, since the legal situation would clearly prevent such a system from being accessible for farmers.

First contact 13.08 Phone call 15.08
We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project. During a phone call, we discussed different parts of our novel approach.
Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system. For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize. Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one. But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany. In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful. Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products. Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant. Upon discussing the specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism. Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.

First Contact: 19.10 Skype Conference 02.10 We approached Tessa Alexanian as a member of the iGEM Human Practice Committee to talk about the value of Human Practice for iGEM and beyond. Moreover, we received a general evaluation of our Human Practice.
We talked about the possible risks from an accidental environmental release as well as rights around the world with regards to the release of GMOs, which is something we worked on in our team team before. Afterwards, we talked about different approaches to regulating scientific research and to transfer it to the field. This gave us a great overview about important topics of Human Practice and gave us examples on how Human Practices can be used.
Furthermore, we talked about judging in general and the interaction with judges at the Giant Jamboree. Tessa also explained to us, that integrating Human Practices into your project is often a question of how your work as changed by the interactions with experts etc.. Human Practice efforts should fit into your overall story, she concluded.

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References

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-							<b style="color:#39F"> Summary </b><br>
+								<b style="color:#39F"> Summary </b><br>
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+								All together, we came into contact with 50+ experts and stakeholders out of more than 18 different countries from all continents.
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+	Their contributions shaped our project into what it is now and the various backgrounds of our experts and their sometimes contradictory opinions elevated our project to a real-world application that aroused interests in farmers, mycologists and experts from the industry alike.
-							<img class="figure image" src="https://2019.igem.org/wiki/images/0/01/T--Bielefeld-CeBiTec--HP_Map_V1.png">
+	Furthermore, we were able to improve biosafety and biosecurity aspects of our project according to the legal situation, guidelines and suggestions for improvements through experts. By doing so, we made sure our Troygenics do not pose any harm for the environment or the consumer.
-						</figure>
+	 <figure class="figure large">
+								<img class="figure image" src="https://2019.igem.org/wiki/images/0/01/T--Bielefeld-CeBiTec--HP_Map_V1.png">
+							</figure>
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 						<h2>Dr Patrick Beuters</h2>
 						<div>He is in the department of Market Development Manager for fungicides in cereals, sugar beet and potatoes.</div>
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 <div>Dr Patrick Beuters gave us insights into the importance of fungicides for agriculture. As a consultant for fungicides for cereals, sugar beet and potatoes he has an extensive overview about all their important products and modes of actions.
 His explanations regarding the history and recent change in usage of fungicides as well as the latest interests in research completed the depictions of Karl Eschenbacher and Bernd Olligs very well.
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 Bernd Olligs is the owner of the farm. He gave us great first-hand insight into the work as a farmer. He showed us how full of meaningful decisions his work is and underlined his statements with stories and anecdotes from his work.
 His depictions of the life as a farmer made us realise how underestimated and unappreciated the work of farmers is in modern society and how complex the issues can be that they are confronted by. They have to assure a maximum yield while minimizing the costs and the impact on the environment while planning years far ahead. This is a task unmatched in modern society.
 During our visit, Bernd Olligs also described the importance of improving the personal contact between farmers and consumers to create a broader understanding of the reasons that drive their decisions and our dependency on their experience.
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+	<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Patrick Beuters</b><br>
+	Market Development Manager for fungicides in cereals, sugar beet and potatoes <br>
+	Bayer CropScience Deutschland GmbH <br>
+	Langenfeld, Germany</div>
+	<hr>
+	Dr. Patrick Beuters supported our project in extensive ways various times. He gave us insights into the usage of fungicides in agriculture and the recent state of research. Based on his suggestions and information about the characteristics and formulations of fungicides, we adapted our project to make our Troygenics applicable with the established methods farmers are already using. Dr. Beuters also consulted us on fungicides, their modes of action and gave us access to two fungicides we integrated into our proof of concept and multiple tests on fungi and self-grown wheat. Also, his suggestions influenced our modelling and he enabled us to visit the Bayer Forward Farm in Rommerskirchen.
+	Furthermore, he forwarded us to a broad range of experts and named us many important sources of information for our project.
+	<div class="content conRight"> <div>
+	First contact 02.07<br>
+	Skype Conference 04.07<br>
+	Extended Phone call 23.08<br>
+	Visit at Bayer Forward Farm in Rommerskirchen 26.09<br>
+	Dr. Patrick Beuters has been one of the most supportive experts during the course of our project. We first approached him at the beginning of July but remained in contact ever since. Since he is responsible for the Fungicide Consulting at Bayer CropScience in Germany and has helped us a lot to receive insights into the economic role of fungicides as well as their overall importance.<br>
+	During our first Skype talk we described our project, our plans as well as our situation. Dr. Beuters then gave us insights into the current situation regarding fungicides and crop protection.<br>
+	He explained, that he would consider plant damaging fungi to be the biggest threat to wheat plants we have to face in Europe. In South America or Africa for example insects are having a larger impact. In Central Europe the harvest loss for wheat through crop damaging fungi is estimated to be between 30 – 60 %.<br>
+	Fungal diseases like powdery mildew (Erysiphe graminis) and leaf blotches (Septoria tritici) are can pose severe threats to cereal plants. And pathogens like Ramularia for barley can have serious impacts on harvests. Furthermore, many fungicides, like Chlorothalonil, are being banned next year due to increasingly stricter regulations and currently, there are no suitable alternatives available.<br>
+	The demand for alternative approaches to fungicides has grown immensely over the last years. Unfortunately, alternatives to fungicides, like biologicals, do not show the same efficiency as fungicides. Because of this the need for alternative approaches is ever increasing.<br>
+	One approach used for the farming of grapes, potatoes and general organic food are fungicides based on copper particles.  However, these can have negative side effects on insects and other ground-dwelling animals and accumulate in the soil.  Luckily as they are not taken up by the plant, they do not pose a threat to the consumer. Currently, agriculture for grapes or potatoes is not feasible in Europe without the application of copper particles. Besides that, copper is a limited resource and there are many other important usages of copper on an industrial scale, for example electronics.<br>
+	Our Troygenic would be almost impossible to apply under current laws and regulations for genetic engineering in Europe. Furthermore, the application of CRISPR outside the laboratory is forbidden in Europe, while it is allowed in, for example, the USA. This also means, that CRISPR cannot be used to increase the resilience of plants against fungi in Europe.<br>
+	Like many other stakeholders, Bayer is investing in research regarding alternatives to fungicides and intensively tries to make biologicals more applicable and a better alternative to common reagents. Of course, this requires close cooperation with farmers, agricultural societies and other stakeholders.<br>
+	In our discussion, we also talked about potential ideas for a cooperation with Bayer. Dr. Beuters forwarded us to a broad range of experts from varies fields, contacts within Bayer and a broad network of farmers.<br>
+	In addition to that, he sent us more research materials to deepen our understanding for our topic even further.<br>
+	Dr. Patrick Beuters explained to us how fungicides are generally applied by farmers. Through his suggestions, we decided to put more thought into adjusting our production system, to make our Troygenics applicable with commonly used methods of applying fungicides. By emulating these methods, our Troygenics would be applicable with the tools farmers already possess. Thereby, the transition from using fungicides to using our Troygenics would facilitate their use by farmers. Since the most common fungicides are used in concentrated liquid form, we adapted our production process to receive a product comparable to the emulsion’s concentrations or concentrations in water that are used to formulate fungicides. After this initial idea, we decided to focus on altering the formulation of our product to resemble exactly the formulation of the most common fungicides. The most widely used fungicides are azoles, since they are broadly applicable.<br>
+	Furthermore, we asked Dr. Beuters for consulting, regarding which kind of fungicide our systems comes close to in its function. Because our Troygenic effects pathogenic fungi directly and therefore prohibits the growth of fungi but also stops the spreading of already existing infections, its mode of action is comparable to the one of most azoles. As a result, Dr. Beuters further consulted us about azoles and their mode of action as well as their formulations and methods of application. In the end, he even gave us access to two fungicides that we used for our proof of concept, some test on self-grown wheat plants and analyses of fungicides.  He also sent us the respective data sheets, manuals and security information as well as further details about safety measures for farmers. He highlighted the importance of the “German Plant Protection Act” (deutsches Pflanzenschutzgesetz) and the “Ordinance on Plant Protection” (Pflanzenschutzsachkundeverordnung).<br>
+	We also called Dr. Beuters to ask for some information to optimize our modelling regarding the development of resistances against fungicides.<br>
+	He pointed out, that since the risk and speed of resistances occurrence is difficult to estimate for a novel mode of action, the risk of resistance development is not easy to include into the development for new fungicides. The threat of resistance development can partially be taken into account for the breeding of resistant plants as an alternative measure to counteract harvest loss. Dr. Beuters forwarded us to a larger set of experts regarding questions for our modelling and data to imbed into it.<br>
+	In general, the risk of resistances being developed is strongly influenced by a sheer endless number of factors. Furthermore, there are many different fungicides with very different modes of action. Farmers try to avoid the progression of resistance development with multiple measures, for example switching fungicides or the order of their usage. Others use mixtures of different reagents. The chambers of agriculture have many suggestions for the usage of fungicides, but at the end farmers are responsible for their own decisions on how to use them, as long as they stick to the law.<br>
+	Besides his extensive support he also gave us the chance to visit the Bayer Forward Farm in Rommerskirchen, a testing area to educate people about agriculture, common methods and recent technological advances. It also resembles a specialised facility for people to get in touch with farmers and stakeholders.<br>
-              <a class="anchor" id="h2"></a>
-					<h1 id=a2 style="margin-top:0px"> General Experts </h1>
-				<hr> <!--Blaue Linie-->
+	</div>
+		</div>
+		</div>
+		<div class="hideDiv">
+			<img class="roundRight" src="https://2019.igem.org/wiki/images/2/2d/T--Bielefeld-CeBiTec--HP_Proffessor_James_Brown.jpg" alt="Bild hoffentlich rund">
+		<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Professor James Brown</b><br>
+	Group Leader – Plant Health<br>
+	John Innes Centre<br>
+	Norwich, United Kingdom</div>
+	<hr style="color:#f1f2f3">
+	Prof. Brown gave us extended insights into the process of fungicide development and approval and commented on various aspects of our project. He advised us on a broad range about things that have to be taken into consideration to make our Troygenics an effective real world application.
+	</button>
+		<div class="content conLeft"> <div> First Contact: 05.09<br>
+	We approached Professor James Brown as an expert for fungal diseases of crops and other plants to receive an evaluation of our project as well as our concept for a potential application.<br>
+	Prof. Brown gave us advice on various points of our project, which he depicted as an interesting approach that should quite certainly be a completely novel approach.<br>
+	First of all, he portrayed that fungicides, although they can be unspecific and have off-target effects, are generally quite specific in their mode of action. Moreover, off-target effects are rather rare and usually weak or hard to detect. Besides this, we would have to put thought into how our system could have similar side effects, he explained. Also, he pointed out that specificity towards a certain pathogen, as well as a broader specificity can both be advantageous. For our system we would have to argue why it would be beneficial to have such a specific mode of action and off-target effects would be important enough to be worth avoiding.<br>
+	Also, he pointed out, that the process of safety testing for fungicides is a rigorous, expensive and time-consuming one. During this process, the fungicides are extensively tested for potential negative impacts for the environment and for humans. This procedure takes nine to ten years after the discovery of the fungicide molecule. These tests are also an important reason, why fungicides generally have very few off-target effects if they are allowed to be commercially used. The patent life of fungicides lasts 17 years. Hence, the companies have only seven to eight years to significantly profit from the sales of a new fungicide while it is still in patent. This period can only be extended by patenting the manufacturing process or keeping important features of the product secret. The process for testing medical pharmaceuticals is quite analogous, although the period of safety testing and is two years shorter, he added. If our Troygenics would be aimed for a commercial use we should put more thought into product marketing and a strategy for IP protection to make sure to make a profit on the system, he suggested.<br>
+	In his view, the short patent life of fungicides is also contributing to the problem of resistance because to make a quick return on their investment, companies have to sell as much of their product as fast as possible. The same effect applies to medical antibiotics, which results in them not being attractive investments for pharma companies anymore. For a commercial application a way to extend the patent life of the Troygenics would thereby be beneficial to maximise the potential profits.<br>
+	If we knew of any possibility that fungi could evolve resistance to our Troygenics this would also have to influence our marketing strategy, Prof. Brown mentioned.<br>
+	Regarding mathematic models he named us two further contacts to come in touch with.<br>
+	One thing Prof. Brown especially pointed out, was that non-specific effects of fungicides are actually often advantageous and thereby desired by farmers because this substitutes multiple treatments with different pesticides. This way a crop can be sprayed just once and still be well protected from multiple diseases. Prof. Brown proposed, that the most important limitation for disease management on the fields is the number of opportunities over the course of a year, when the ground is dry enough for the usage of a tractor but not to dry, so plants are not getting damaged because of the drought. Only then, it is possible to spray against crop diseases of local importance. The conditions to do this are really fairly specific. There should be no rain at the moment and no rain expected for the next 12 to 14 hours at wind speed forces of 1-3.
+	For crops in greenhouses, there is more flexibility, but the number of sprays is aimed to be minimized because of two reasons. Firstly, the cost of spraying, the staff and the fuel should be minimized. And secondly, crops with fewer treatments of reagents like fungicides are more attractive for supermarkets. Prof. Brown also gave a distinct for this development: During the 34 years of his career the average number of sprays applied per year to blackcurrants, an important crop where he lives, dropped from 37 to one.<br>
+	Moreover, there has been concerns about negative, non-specific effects of fungicides on soil microbiota but far to less research on the subject. Prof. Brown portrayed, that he would consider a larger effect of the fungicides very unlikely. The amount of fungicides sprayed per unit area is very low. Furthermore, fungicides are usually applied when the crop canopy is well-formed and the great majority of the spray lands on the plant.<br>
+	While there are some advantageous in having a very specific targeted method to fight certain crop pathogens, there are also define disadvantageous. It is important to know when such a system would be beneficial and when it would not be, Prof. Brown concluded.<br>
+	The biggest current concern, regarding the off-target effects of fungicides is that triazole fungicides, which are targeting ERG11 (CYP51) in the ergosterol synthesis pathway in fungi, can have off-target effects on at least one enzyme involved in the hormone synthesis in mammals. These effects are still very small though and far below the level detectable by epidemiological analysis. The EU legislation still requires strictly no off-target effects on the mammalian reproductive system, by whatever method of testing.<br>
+	This behaviour has made the companies, that develop new fungicides, very nervous, since the increasing threat of losing huge amounts of invested money because of unknown off-target effects. Thereby, a system like ours would have to prove that it does not have any off-target biological and biochemical processes, especially regarding the mammalian reproductive system in any way.<br>
+	Since the interest in fungicide analogues is quite high, a range of methods have been suggested. But these are most often not as effective as the fungicides and for example require multiple applications on the field. Also transporting and application cost would increase.<br>
+	Further very important characteristics of our method would be its efficacy and therefore the likely cost of the application. Additionally, a reinfection of the plant after the application should be expected. Also, it would be important to know how long our Troygenics would take to degrade and thereby how many applications would be necessary to effectively protect the crop.<br>
+</div>
+	</div>
+			</div>
-				<div class="hideDiv">
-					<img class="roundLeft" src="https://2019.igem.org/wiki/images/4/46/T--Bielefeld-CeBiTec--HP_Beuters.jpg">
-				<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Patrick Beuters</b><br>
-				Market Development Manager for fungicides in cereals, sugar beet and potatoes <br>
-				Bayer CropScience Deutschland GmbH <br>
-				Langenfeld, Germany</div>
-				<hr>
-				Dr. Patrick Beuters supported our project in extensive ways various times. He gave us insights into the usage of fungicides in agriculture and the recent state of research. Based on his suggestions and information about the characteristics and formulations of fungicides, we adapted our project to make our Troygenics applicable with the established methods farmers are already using. Dr. Beuters also consulted us on fungicides, their modes of action and gave us access to two fungicides we integrated into our proof of concept and multiple tests on fungi and self-grown wheat. Also, his suggestions influenced our modelling and he enabled us to visit the Bayer Forward Farm in Rommerskirchen.
-				Furthermore, he forwarded us to a broad range of experts and named us many important sources of information for our project.
-				<div class="content conRight"> <div>
-				First contact 02.07<br>
-				Skype Conference 04.07<br>
-				Extended Phone call 23.08<br>
-				Visit at Bayer Forward Farm in Rommerskirchen 26.09<br>
-				Dr. Patrick Beuters has been one of the most supportive experts during the course of our project. We first approached him at the beginning of July but remained in contact ever since. Since he is responsible for the Fungicide Consulting at Bayer CropScience in Germany and has helped us a lot to receive insights into the economic role of fungicides as well as their overall importance.<br>
-				During our first Skype talk we described our project, our plans as well as our situation. Dr. Beuters then gave us insights into the current situation regarding fungicides and crop protection.<br>
-				He explained, that he would consider plant damaging fungi to be the biggest threat to wheat plants we have to face in Europe. In South America or Africa for example insects are having a larger impact. In Central Europe the harvest loss for wheat through crop damaging fungi is estimated to be between 30 – 60 %.<br>
-				Fungal diseases like powdery mildew (Erysiphe graminis) and leaf blotches (Septoria tritici) are can pose severe threats to cereal plants. And pathogens like Ramularia for barley can have serious impacts on harvests. Furthermore, many fungicides, like Chlorothalonil, are being banned next year due to increasingly stricter regulations and currently, there are no suitable alternatives available.<br>
-				The demand for alternative approaches to fungicides has grown immensely over the last years. Unfortunately, alternatives to fungicides, like biologicals, do not show the same efficiency as fungicides. Because of this the need for alternative approaches is ever increasing.<br>
-				One approach used for the farming of grapes, potatoes and general organic food are fungicides based on copper particles.  However, these can have negative side effects on insects and other ground-dwelling animals and accumulate in the soil.  Luckily as they are not taken up by the plant, they do not pose a threat to the consumer. Currently, agriculture for grapes or potatoes is not feasible in Europe without the application of copper particles. Besides that, copper is a limited resource and there are many other important usages of copper on an industrial scale, for example electronics.<br>
-				Our Troygenic would be almost impossible to apply under current laws and regulations for genetic engineering in Europe. Furthermore, the application of CRISPR outside the laboratory is forbidden in Europe, while it is allowed in, for example, the USA. This also means, that CRISPR cannot be used to increase the resilience of plants against fungi in Europe.<br>
-				Like many other stakeholders, Bayer is investing in research regarding alternatives to fungicides and intensively tries to make biologicals more applicable and a better alternative to common reagents. Of course, this requires close cooperation with farmers, agricultural societies and other stakeholders.<br>
-				In our discussion, we also talked about potential ideas for a cooperation with Bayer. Dr. Beuters forwarded us to a broad range of experts from varies fields, contacts within Bayer and a broad network of farmers.<br>
-				In addition to that, he sent us more research materials to deepen our understanding for our topic even further.<br>
-				Dr. Patrick Beuters explained to us how fungicides are generally applied by farmers. Through his suggestions, we decided to put more thought into adjusting our production system, to make our Troygenics applicable with commonly used methods of applying fungicides. By emulating these methods, our Troygenics would be applicable with the tools farmers already possess. Thereby, the transition from using fungicides to using our Troygenics would facilitate their use by farmers. Since the most common fungicides are used in concentrated liquid form, we adapted our production process to receive a product comparable to the emulsion’s concentrations or concentrations in water that are used to formulate fungicides. After this initial idea, we decided to focus on altering the formulation of our product to resemble exactly the formulation of the most common fungicides. The most widely used fungicides are azoles, since they are broadly applicable.<br>
-				Furthermore, we asked Dr. Beuters for consulting, regarding which kind of fungicide our systems comes close to in its function. Because our Troygenic effects pathogenic fungi directly and therefore prohibits the growth of fungi but also stops the spreading of already existing infections, its mode of action is comparable to the one of most azoles. As a result, Dr. Beuters further consulted us about azoles and their mode of action as well as their formulations and methods of application. In the end, he even gave us access to two fungicides that we used for our proof of concept, some test on self-grown wheat plants and analyses of fungicides.  He also sent us the respective data sheets, manuals and security information as well as further details about safety measures for farmers. He highlighted the importance of the “German Plant Protection Act” (deutsches Pflanzenschutzgesetz) and the “Ordinance on Plant Protection” (Pflanzenschutzsachkundeverordnung).<br>
-				We also called Dr. Beuters to ask for some information to optimize our modelling regarding the development of resistances against fungicides.<br>
-				He pointed out, that since the risk and speed of resistances occurrence is difficult to estimate for a novel mode of action, the risk of resistance development is not easy to include into the development for new fungicides. The threat of resistance development can partially be taken into account for the breeding of resistant plants as an alternative measure to counteract harvest loss. Dr. Beuters forwarded us to a larger set of experts regarding questions for our modelling and data to imbed into it.<br>
-				In general, the risk of resistances being developed is strongly influenced by a sheer endless number of factors. Furthermore, there are many different fungicides with very different modes of action. Farmers try to avoid the progression of resistance development with multiple measures, for example switching fungicides or the order of their usage. Others use mixtures of different reagents. The chambers of agriculture have many suggestions for the usage of fungicides, but at the end farmers are responsible for their own decisions on how to use them, as long as they stick to the law.<br>
-				Besides his extensive support he also gave us the chance to visit the Bayer Forward Farm in Rommerskirchen, a testing area to educate people about agriculture, common methods and recent technological advances. It also resembles a specialised facility for people to get in touch with farmers and stakeholders.<br>
+			<div class="hideDiv">
+				<img class="roundLeft" src="https://2019.igem.org/wiki/images/e/ec/T--Bielefeld-CeBiTec--HP_Francismar_Marcelino-Guimaraes.jpg">
+			<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dra. Francismar Corrêa Marcelino-Guimaraes</b><br>
+	Research and Development <br>
+	The Brazilian Agricultural Research Corporation (Embrapa) <br>
+	Londrina, Brasil</div>
+	<hr>
+	Dra. Marcelino-Guimaraes helped us to grasp the impact of the Asian Soybean Rust, which would be an exceptional good target for our Troygenics. She also depicted work and the current state of research regarding the Asian Soybean Rust.
+			<div class="content conRight"> <div>
+				First Contact 19.09
+	We reached out to Dra. Francismar Corrêa Marcelino-Guimaraes as a contact at The Brazilian Agricultural Research Corporation (Embrapa) to ask her about the most important fungal crop pathogens in Brazil and South America, especially the Asian Soybean Rust.
+	Dra. Marcelino-Guimaraes confirmed the Asian Soybean Rust to be the most important crop pathogen in Brazil and depicted the current situation: All strategies to control this pathogen have failed and the resistance to fungicides is continuously increasing. Moreover, genetic resistance, referring to the resistance genes from Soybean, are not effective against all variants of the pathogen that occur on the field. The fungus that is causing the Asians Soybean Rust (Phakopsora pachyrhizi) is a biotrophic pathogen. Because of this, it can not be grown in artificial culture media. As a result, the transformation is very complicated and there is no protocol available.
+	Dra. Marcelino-Guimaraes is also part of an International Consortium with the objective of obtaining a reference genome of this pathogen. The genome should be public this year (2019) with a set of all 22000 predicted genes.
+	The Asian Soybean Rust is the only crop disease, single pathogen targeted fungicides are being used for because of the massive impact of this crop harming fungus in Asia, Africa and South America. This makes this fungal pathogen an exceptional good target for the application of our Troygenics.
+	Dra. Marcelino-Guimaraes even pointed out, that we are taking part in really important research for the scientific community.
+	She also forwarded us to colleagues of her and named us further sources of information about the impact of the Asian Soybean Rust and the current state of research.
+	</div>
 				</div>
-					</div>
-					</div>
-					<div class="hideDiv">
-						<img class="roundRight" src="https://2019.igem.org/wiki/images/2/2d/T--Bielefeld-CeBiTec--HP_Proffessor_James_Brown.jpg" alt="Bild hoffentlich rund">
-					<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Professor James Brown</b><br>
-				Group Leader – Plant Health<br>
-				John Innes Centre<br>
-				Norwich, United Kingdom</div>
-				<hr style="color:#f1f2f3">
-				Prof. Brown gave us extended insights into the process of fungicide development and approval and commented on various aspects of our project. He advised us on a broad range about things that have to be taken into consideration to make our Troygenics an effective real world application.
-				</button>
-					<div class="content conLeft"> <div> First Contact: 05.09<br>
-				We approached Professor James Brown as an expert for fungal diseases of crops and other plants to receive an evaluation of our project as well as our concept for a potential application.<br>
-				Prof. Brown gave us advice on various points of our project, which he depicted as an interesting approach that should quite certainly be a completely novel approach.<br>
-				First of all, he portrayed that fungicides, although they can be unspecific and have off-target effects, are generally quite specific in their mode of action. Moreover, off-target effects are rather rare and usually weak or hard to detect. Besides this, we would have to put thought into how our system could have similar side effects, he explained. Also, he pointed out that specificity towards a certain pathogen, as well as a broader specificity can both be advantageous. For our system we would have to argue why it would be beneficial to have such a specific mode of action and off-target effects would be important enough to be worth avoiding.<br>
-				Also, he pointed out, that the process of safety testing for fungicides is a rigorous, expensive and time-consuming one. During this process, the fungicides are extensively tested for potential negative impacts for the environment and for humans. This procedure takes nine to ten years after the discovery of the fungicide molecule. These tests are also an important reason, why fungicides generally have very few off-target effects if they are allowed to be commercially used. The patent life of fungicides lasts 17 years. Hence, the companies have only seven to eight years to significantly profit from the sales of a new fungicide while it is still in patent. This period can only be extended by patenting the manufacturing process or keeping important features of the product secret. The process for testing medical pharmaceuticals is quite analogous, although the period of safety testing and is two years shorter, he added. If our Troygenics would be aimed for a commercial use we should put more thought into product marketing and a strategy for IP protection to make sure to make a profit on the system, he suggested.<br>
-				In his view, the short patent life of fungicides is also contributing to the problem of resistance because to make a quick return on their investment, companies have to sell as much of their product as fast as possible. The same effect applies to medical antibiotics, which results in them not being attractive investments for pharma companies anymore. For a commercial application a way to extend the patent life of the Troygenics would thereby be beneficial to maximise the potential profits.<br>
-				If we knew of any possibility that fungi could evolve resistance to our Troygenics this would also have to influence our marketing strategy, Prof. Brown mentioned.<br>
-				Regarding mathematic models he named us two further contacts to come in touch with.<br>
-				One thing Prof. Brown especially pointed out, was that non-specific effects of fungicides are actually often advantageous and thereby desired by farmers because this substitutes multiple treatments with different pesticides. This way a crop can be sprayed just once and still be well protected from multiple diseases. Prof. Brown proposed, that the most important limitation for disease management on the fields is the number of opportunities over the course of a year, when the ground is dry enough for the usage of a tractor but not to dry, so plants are not getting damaged because of the drought. Only then, it is possible to spray against crop diseases of local importance. The conditions to do this are really fairly specific. There should be no rain at the moment and no rain expected for the next 12 to 14 hours at wind speed forces of 1-3.
-				For crops in greenhouses, there is more flexibility, but the number of sprays is aimed to be minimized because of two reasons. Firstly, the cost of spraying, the staff and the fuel should be minimized. And secondly, crops with fewer treatments of reagents like fungicides are more attractive for supermarkets. Prof. Brown also gave a distinct for this development: During the 34 years of his career the average number of sprays applied per year to blackcurrants, an important crop where he lives, dropped from 37 to one.<br>
-				Moreover, there has been concerns about negative, non-specific effects of fungicides on soil microbiota but far to less research on the subject. Prof. Brown portrayed, that he would consider a larger effect of the fungicides very unlikely. The amount of fungicides sprayed per unit area is very low. Furthermore, fungicides are usually applied when the crop canopy is well-formed and the great majority of the spray lands on the plant.<br>
-				While there are some advantageous in having a very specific targeted method to fight certain crop pathogens, there are also define disadvantageous. It is important to know when such a system would be beneficial and when it would not be, Prof. Brown concluded.<br>
-				The biggest current concern, regarding the off-target effects of fungicides is that triazole fungicides, which are targeting ERG11 (CYP51) in the ergosterol synthesis pathway in fungi, can have off-target effects on at least one enzyme involved in the hormone synthesis in mammals. These effects are still very small though and far below the level detectable by epidemiological analysis. The EU legislation still requires strictly no off-target effects on the mammalian reproductive system, by whatever method of testing.<br>
-				This behaviour has made the companies, that develop new fungicides, very nervous, since the increasing threat of losing huge amounts of invested money because of unknown off-target effects. Thereby, a system like ours would have to prove that it does not have any off-target biological and biochemical processes, especially regarding the mammalian reproductive system in any way.<br>
-				Since the interest in fungicide analogues is quite high, a range of methods have been suggested. But these are most often not as effective as the fungicides and for example require multiple applications on the field. Also transporting and application cost would increase.<br>
-				Further very important characteristics of our method would be its efficacy and therefore the likely cost of the application. Additionally, a reinfection of the plant after the application should be expected. Also, it would be important to know how long our Troygenics would take to degrade and thereby how many applications would be necessary to effectively protect the crop.<br>
 				</div>
-						</div>
+				<div class="hideDiv">
+					<img class="roundRight" src="https://2019.igem.org/wiki/images/c/c7/T--Bielefeld-CeBiTec--HP_Zabel.png" alt="Bild hoffentlich rund">
+				<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Paul Zabel</b><br>
+	Research Associate at DLR working on the EDEN ISS project<br>
+	German Aerospace Center (DLR)<br>
+	Bremen, Germany (project: Antarctica)</div>
+	<hr style="color:#f1f2f3">
+	Paul Zabel gave us insights into the impact of fungi under the unique conditions of an experimental project with the objective to grow crops inside a specialized greenhouse in Antarctica. The pilot project EDEN ISS was established by the German Aerospace Center to test systems for plant cultivation during space missions.
+	Beyond that, we discussed our Troygenics as a potential application on space missions, since the targeted and independent biological production could have severe benefits for the self-sustainability of space colonies.
-						<div class="hideDiv">
+	</button>
-							<img class="roundLeft" src="https://2019.igem.org/wiki/images/e/ec/T--Bielefeld-CeBiTec--HP_Francismar_Marcelino-Guimaraes.jpg">
+				<div class="content conLeft"> <div> First contact 09.09
-						<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dra. Francismar Corrêa Marcelino-Guimaraes</b><br>
+	Phone call 10.09
-				Research and Development <br>
+	Video conference 13.09
-				The Brazilian Agricultural Research Corporation (Embrapa) <br>
+	Paul Zabel works at a Research Associate for the German Aerospace Center (DLR) in the Space Segment Systems Analysis department and has been part of a unique pilot experiment, the EDEN ISS project. The objective of this project, founded by the European Commission under the Horizon 2020 research programme, was to test a special laboratory suited for plant breeding and food production on future space missions. To test this laboratory under extreme conditions Paul Zabel spent one year in Antarctica near the AWI's Neumayer Station III to cultivate vegetables and herbs.<br>
-				Londrina, Brasil</div>
+	We approached him to receive an evaluation on the impact of fungi under these special conditions, as well as the precautions that have to be taken to prevent plant damaging fungi.<br>
-				<hr>
+	After we called Paul Zabel, we organised a video conference to discuss the role of fungi during his expedition and some aspects of our project.<br>
-				Dra. Marcelino-Guimaraes helped us to grasp the impact of the Asian Soybean Rust, which would be an exceptional good target for our Troygenics. She also depicted work and the current state of research regarding the Asian Soybean Rust.
+	There have been two shifts on the EDEN ISS since the start of the project, one in 2018 and one in 2019. A season lasts from February to November. Paul Zabel spent the very first season at the South Pole for this project.<br>
-						<div class="content conRight"> <div>
+	In both years, there have been problems with fungi on the station. During the first months of its use the fungi display a faster growth and can form biofilms in the nutrient solutions of the plants. Under these conditions, fungi can start to grow on moist parts of pipes and uneven surfaces. Over time the growth of the fungi reaches a point of self-regulation. During both years, chlorine cleaner was used to counteract this problem with only moderate success. In the next years, the usage of such methods is planned to be reduced to a minimum. Samples from plant surfaces and the water have been taken and are being analyzed by the Astrobiology Group of the Institute of Aerospace Medicine in Cologne. Still the general burden of fungi on vegetables produced in the Antarctic has shown to be 1000 – 10000 times smaller than the one of store bought vegetables. Analyzed contamination samples at the arctic greenhouse rather contained spores of fungi than bacteria.<br>
-							First Contact 19.09
+	For the plants, these fungi do not pose severe threats, only the optics of some plants are affected by the fungi. But beyond that, pipes and filters can become clogged through the biofilms and can cause technical issues. Also, the plants are growing in special cubes out of mineral wool that can become overgrown by fungi. This can potentially lead to the stem of the plants to soften up. As a result, the stems of healthy plants can break and the plant dies. Still this issue only affects a small number of plants per year.<br>
-				We reached out to Dra. Francismar Corrêa Marcelino-Guimaraes as a contact at The Brazilian Agricultural Research Corporation (Embrapa) to ask her about the most important fungal crop pathogens in Brazil and South America, especially the Asian Soybean Rust.
+	At the moment, the seeds are not being sterilized or treated in any special way, since most of the surroundings are clean surfaces, also the cubes of mineral wool are being heat-treated before use.<br>
-				Dra. Marcelino-Guimaraes confirmed the Asian Soybean Rust to be the most important crop pathogen in Brazil and depicted the current situation: All strategies to control this pathogen have failed and the resistance to fungicides is continuously increasing. Moreover, genetic resistance, referring to the resistance genes from Soybean, are not effective against all variants of the pathogen that occur on the field. The fungus that is causing the Asians Soybean Rust (Phakopsora pachyrhizi) is a biotrophic pathogen. Because of this, it can not be grown in artificial culture media. As a result, the transformation is very complicated and there is no protocol available.
+	It is still under discussion, how extensive the sterilization process for later space missions would have to be. Since, on the International Space Station (ISS), everything is mandatory being sterilized and there are still fungal contaminations occurring in space, Zabel reports. Besides that, growing plants can not be sterilized. In the Antarctic, people are visiting the greenhouse with protective clothes, are not allowed to touch anything and have to follow certain rules like “no food” as an “intermediate solution”. This approach could be adapted for space missions later on. Cleaning and sterilization procedures are time-consuming and expensive and will be avoided if possible. In addition to this, vapors of cleaning agents would have to be filtered out in space.<br>
-				Dra. Marcelino-Guimaraes is also part of an International Consortium with the objective of obtaining a reference genome of this pathogen. The genome should be public this year (2019) with a set of all 22000 predicted genes.
+	The question arises, if a sterilization of a garden in space would even be possible. For example, on the ISS, four salads have been grown, sterilized with chlorine solution and water and eaten. But it is a rather minor effort to sterilize four salads. The greenhouse Pail Zabel worked in has a base area of 12,5 m2 and would be almost impossible to completely sanitize. Moreover, the water consumption of the sterilization process is quite high. To sterilize 1,5 kg of rocket (salad) around 60-70 l of water are needed, although the salads shelf life expands for about one or two weeks treated this way. So, it has to be evaluated, if the sterilization process would be worth all of this and if the approach sterilizing everything on space missions could actually be continued on this level. After all, the vegetables grown in space could still be eaten without any sterilization process. Maybe the rules on this have to be loosened a little bit, Paul Zabel concludes.<br>
-				The Asian Soybean Rust is the only crop disease, single pathogen targeted fungicides are being used for because of the massive impact of this crop harming fungus in Asia, Africa and South America. This makes this fungal pathogen an exceptional good target for the application of our Troygenics.
+	Tests have been conducted with ozone lead into the nutrient solution to lower the growth of fungi. But this has led to the binding and flocculation of nutrients like Iron or Calcium, making them inaccessible for the plants. For future mission in the Antarctic, further measures are already planned. Like tests regarding the sterilization of the seeds and the usage of hydrogen peroxide solution with silver ions added. Also, biological approaches are planned to promote a natural balance and the usage of other strains of fungi to keep the harmful ones at bay could also be a potential measure.<br>
-				Dra. Marcelino-Guimaraes even pointed out, that we are taking part in really important research for the scientific community.
+	One thing Paul Zabel also pointed out, was that the need for the sterilization of the plants would considerably limit the selection of the plants, since the plant surface has to be accessible for the fungicide. While a salad is quite easy to sterilize, this is almost impossible for crops like carrots or potatoes because they need to be in direct contact with the nutrient solution. Research on soil-free cultivation methods of these plants is being conducted but showed to be rather challenging.
-				She also forwarded us to colleagues of her and named us further sources of information about the impact of the Asian Soybean Rust and the current state of research.
+	Keeping the current situation of plant breeding in space in mind, there could actually be an application of our Troygenics for future space missions. Since the ultimate goal of a colony on, for example, Mars would be the total self-sufficiency of the colony, the supply on reagents to fight plant pathogens would be extremely limited. A possibility to produce targeted reagents, that only work on the fungi that are harming the plant without harming any of the beneficial fungi for the plant or the plant itself could be a huge benefit. Furthermore, our Troygenics could be modified, adapted and produced in a laboratory in space using E. coli. As a result, the colony could operate independent from supplies from earth. Since the dependency on the own harvest would be way more important in a self-sufficient space colony and the loss of plants through pathogens, fungi, or even radiation in space should be limited to an absolute minimum, our system could contribute to ensure the protection of crops under these conditions. Avoiding technical difficulties, like clogged pipes or filters would be another important issue, since repair parts are often not available.<br>
+	The ability to manufacture tools or measures to solve problems, like the Troygenics, to assure the self-sufficiency of the colony in space is extremely important, Paul Zabel resumes.<br>
-				</div>
+</div>
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-							<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Paul Zabel</b><br>
-				Research Associate at DLR working on the EDEN ISS project<br>
-				German Aerospace Center (DLR)<br>
-				Bremen, Germany (project: Antarctica)</div>
-				<hr style="color:#f1f2f3">
-				Paul Zabel gave us insights into the impact of fungi under the unique conditions of an experimental project with the objective to grow crops inside a specialized greenhouse in Antarctica. The pilot project EDEN ISS was established by the German Aerospace Center to test systems for plant cultivation during space missions.
-				Beyond that, we discussed our Troygenics as a potential application on space missions, since the targeted and independent biological production could have severe benefits for the self-sustainability of space colonies.
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-							<div class="content conLeft"> <div> First contact 09.09
-				Phone call 10.09
-				Video conference 13.09
-				Paul Zabel works at a Research Associate for the German Aerospace Center (DLR) in the Space Segment Systems Analysis department and has been part of a unique pilot experiment, the EDEN ISS project. The objective of this project, founded by the European Commission under the Horizon 2020 research programme, was to test a special laboratory suited for plant breeding and food production on future space missions. To test this laboratory under extreme conditions Paul Zabel spent one year in Antarctica near the AWI's Neumayer Station III to cultivate vegetables and herbs.<br>
-				We approached him to receive an evaluation on the impact of fungi under these special conditions, as well as the precautions that have to be taken to prevent plant damaging fungi.<br>
-				After we called Paul Zabel, we organised a video conference to discuss the role of fungi during his expedition and some aspects of our project.<br>
-				There have been two shifts on the EDEN ISS since the start of the project, one in 2018 and one in 2019. A season lasts from February to November. Paul Zabel spent the very first season at the South Pole for this project.<br>
-				In both years, there have been problems with fungi on the station. During the first months of its use the fungi display a faster growth and can form biofilms in the nutrient solutions of the plants. Under these conditions, fungi can start to grow on moist parts of pipes and uneven surfaces. Over time the growth of the fungi reaches a point of self-regulation. During both years, chlorine cleaner was used to counteract this problem with only moderate success. In the next years, the usage of such methods is planned to be reduced to a minimum. Samples from plant surfaces and the water have been taken and are being analyzed by the Astrobiology Group of the Institute of Aerospace Medicine in Cologne. Still the general burden of fungi on vegetables produced in the Antarctic has shown to be 1000 – 10000 times smaller than the one of store bought vegetables. Analyzed contamination samples at the arctic greenhouse rather contained spores of fungi than bacteria.<br>
-				For the plants, these fungi do not pose severe threats, only the optics of some plants are affected by the fungi. But beyond that, pipes and filters can become clogged through the biofilms and can cause technical issues. Also, the plants are growing in special cubes out of mineral wool that can become overgrown by fungi. This can potentially lead to the stem of the plants to soften up. As a result, the stems of healthy plants can break and the plant dies. Still this issue only affects a small number of plants per year.<br>
-				At the moment, the seeds are not being sterilized or treated in any special way, since most of the surroundings are clean surfaces, also the cubes of mineral wool are being heat-treated before use.<br>
-				It is still under discussion, how extensive the sterilization process for later space missions would have to be. Since, on the International Space Station (ISS), everything is mandatory being sterilized and there are still fungal contaminations occurring in space, Zabel reports. Besides that, growing plants can not be sterilized. In the Antarctic, people are visiting the greenhouse with protective clothes, are not allowed to touch anything and have to follow certain rules like “no food” as an “intermediate solution”. This approach could be adapted for space missions later on. Cleaning and sterilization procedures are time-consuming and expensive and will be avoided if possible. In addition to this, vapors of cleaning agents would have to be filtered out in space.<br>
-				The question arises, if a sterilization of a garden in space would even be possible. For example, on the ISS, four salads have been grown, sterilized with chlorine solution and water and eaten. But it is a rather minor effort to sterilize four salads. The greenhouse Pail Zabel worked in has a base area of 12,5 m2 and would be almost impossible to completely sanitize. Moreover, the water consumption of the sterilization process is quite high. To sterilize 1,5 kg of rocket (salad) around 60-70 l of water are needed, although the salads shelf life expands for about one or two weeks treated this way. So, it has to be evaluated, if the sterilization process would be worth all of this and if the approach sterilizing everything on space missions could actually be continued on this level. After all, the vegetables grown in space could still be eaten without any sterilization process. Maybe the rules on this have to be loosened a little bit, Paul Zabel concludes.<br>
-				Tests have been conducted with ozone lead into the nutrient solution to lower the growth of fungi. But this has led to the binding and flocculation of nutrients like Iron or Calcium, making them inaccessible for the plants. For future mission in the Antarctic, further measures are already planned. Like tests regarding the sterilization of the seeds and the usage of hydrogen peroxide solution with silver ions added. Also, biological approaches are planned to promote a natural balance and the usage of other strains of fungi to keep the harmful ones at bay could also be a potential measure.<br>
-				One thing Paul Zabel also pointed out, was that the need for the sterilization of the plants would considerably limit the selection of the plants, since the plant surface has to be accessible for the fungicide. While a salad is quite easy to sterilize, this is almost impossible for crops like carrots or potatoes because they need to be in direct contact with the nutrient solution. Research on soil-free cultivation methods of these plants is being conducted but showed to be rather challenging.
-				Keeping the current situation of plant breeding in space in mind, there could actually be an application of our Troygenics for future space missions. Since the ultimate goal of a colony on, for example, Mars would be the total self-sufficiency of the colony, the supply on reagents to fight plant pathogens would be extremely limited. A possibility to produce targeted reagents, that only work on the fungi that are harming the plant without harming any of the beneficial fungi for the plant or the plant itself could be a huge benefit. Furthermore, our Troygenics could be modified, adapted and produced in a laboratory in space using E. coli. As a result, the colony could operate independent from supplies from earth. Since the dependency on the own harvest would be way more important in a self-sufficient space colony and the loss of plants through pathogens, fungi, or even radiation in space should be limited to an absolute minimum, our system could contribute to ensure the protection of crops under these conditions. Avoiding technical difficulties, like clogged pipes or filters would be another important issue, since repair parts are often not available.<br>
-				The ability to manufacture tools or measures to solve problems, like the Troygenics, to assure the self-sufficiency of the colony in space is extremely important, Paul Zabel resumes.<br>
+		</div>
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-								<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Gopaljee Jha</b><br>
-								PhD. student <br>
-								National Institute of Plant Genome Research - NIPGR<br>
-								New Delhi, India</div>
-								<hr>
-								Gopaljee Jha furthered our research and influenced some of our decisions and sparked new ideas on how to induce endocytosis in fungi. Additionally, he gave us some insights into the legal situation of genetic engineering in India.
-								<div class="content conRight"> <div>
-									First Contact 05.09
-				Skype Conference 06.09
-				Gopaljee Jha is doing his PhD. At the National Institute of Plant Genome Research (NIPGR) in New Delhi and focused his Research on plant microbe interactions.<br>
-				After we presented our project and our situation, we discussed several aspects of our work.<br>
-				He suggested us to alternatively think about modifying mycoviruses for our project, which could be a focus of later research.<br>
-				He pointed out, that special bacteria, called Burkholderia, are able to express proteins which can destroy the cell walls of fungi. As far as they were researched, this worked in every fungus that was tested.<br>
-				Additionally, he mentioned that fungi are also capable of quorum sensing, like bacteria. Because of this, he expected hormones to be a better possibility to induce endocytosis. At this point, we already decided to use mating factors for this purpose and have been affirmed by this advice.<br>
-				Gopaljee Jha underlined, that the cell walls of yeasts and fungi differ in many ways and tests with yeast are thereby not representative for the functionality in fungi. This was reflected in our proof of concept, by introducing Aspergilli as filamentous fungi as a second organism to test our system in.<br>
-				In general, Gopaljee Jha assessed specificity through endocytosis to be difficult to achieve, since many fungi are very closely related.<br>
-				As far as he knew, there is no gene that is expressed by all pathogenic fungi during infection but expected that we should be able to find some via RNA analysis if we put enough effort into it.<br>
-				Regarding the legal situation of genetic engineering in India, Gopaljee Jha stated, that because India is located between the USA and the EU its market is influenced by both. As a result, transgenic organisms would be almost impossible to set free under legal conditions. To some extent, specially breed variations of crops should be possible, though. At least as long as they are interspecific.<br>
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-									<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Karl-Heinz Kogel</b><br>
-								Head of the institute for Phytopathology<br>
-				University of Giessen<br>
-								Giessen, Germany</div>
-								<hr style="color:#f1f2f3">
-								We approached Prof. Dr. Karl-Heinz Kogel as an expert of Phytopathology to receive further information about important fungal crop pathogens of economic importance. During a call he advised us on a range of pathogenic fungi and potential concepts of fighting them by implementing latest research into our project.
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-									<div class="content conLeft"> <div> First contact 25.06<br>
-				Phone call 19.08<br>
-				We contacted Prof. Dr. Karl-Heinz Kogel, head of the institute for phytopathology at the University of Giessen (Germany), to gather additional information on fungal crop pathogens, their impact and their ecological and economic value.<br>
-				After we explained what the iGEM competition is and our project we discussed the different parts of our work.<br>
-				Prof. Kogel pointed out, that our project has to be planned with a lot of foresight, since the current political and social opinion on genetic engineering would prevent such an application in the near future. With the legal situation regarding this topic in the EU he did not expect a system like our Troygenics to be applicable anytime soon.<br>
-				Besides that, he still liked our approach and considered it a creative idea. Although, he also mentioned, that most comparable approaches do not try to solve this many problems at once, which makes our project susceptible to more problems in direct comparison.<br>
-				He was especially interested in our approach of an induced endocytic uptake into the pathogenic fungi via specific ligands on the surface of the Troygenics.<br>
-				As an additional, similar approach he proposed the idea of using only dsRNA instead of the Troygenics and gene silencing instead of CRISPR/ Cas. While this concept would elute some of the problems of our system, like the required uptake of, in comparison, larger particles	into the target cell, it would also establish new ones. On one hand this would prevent GMOs to form on the fields. This would make it more realistic to implement and is closer to the current praxis. On the other hand, this would require the use of siRNAs which are distinctly more instable and way larger amounts of them would be needed to achieve the same effect. Moreover, this alternative approach would lack the shuttle our concept provides. Finally, the biological production of the Troygenics via E. coli would be a clear advantage in direct comparison.<br>
-				Although, this was a great suggestion, we still considered our approach as a better solution to generate a broader range of potential applications.<br>
-				Furthermore, Prof. Kogel gave us additional information about pathogenic crop fungi and their impact on agriculture. As a further potential target, that would be important to come by, he named Fusarium graminearum as an important pest of wheat and corn plants all around the world. He even mentioned some genes of certain pathways to target for our system. In this case, the pathogen usually is treated with azole-based pesticides, which deactivate proteins essential for the infection of the plant.<br> F. graminearum has become a substantial threat to the food security of the named crops.<br>
-				Regarding Puccinia graminis, a devastating wheat pathogen, that managed to develop multiresistant strains and is threatening the food supply in parts of Africa and that is spreading continuously, he confirmed the danger of the pathogen and added, that its impact is going to increase dramatically because of the progressing climate change.<br>
-				Prof. Kogel also confirmed, that problems due to fungicide resistance are already a relevant threat to our food security and supply. A far range of papers are published about this topic.<br>
-				At the end, we asked Prof. Kogel about methods to distinguish between different spores of fungi and to determine their species without sequencing parts of their genome. Prof. Kogel explained, that the morphology of the spores of different fungal species are quite easy to distinguish with a closer investigation via microscopy.<br>
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-										<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Wei Xiong </b><br>
-										Crop Modeler and Physiologist at CIMMYT’s Sustainable Intensification Program <br>
-										International Maize and Wheat Improvement Center – CIMMYT, Chinese Academy of Agricultural Sciences <br>
-										Beijing, P.R. China </div>
-										<hr>
-										Wei Xiong furthered our knowledge about how a mathematic model for the development of fungal resistances can be established and which data sets this would require. He also gave us insights into where these models are used, how important they are and gave us examples how they could be implemented into measures to protect food production.<br>
-										<div class="content conRight"> <div>
-											First Contact 21.09
-				We reached out to Wei Xiong as a member of the International Maize and Wheat Improvement Center (CIMMYT) in China after we read about his former research, investigating in the influences of weather conditions on harvest in Eastern Europe.<br>
-				We asked him about how changes of the weather and the influence of climate change is estimated to impact the crop production in Europe. While his work only not considered the influence of crop pathogens, but he stated, recent research estimated that the magnitude of the effects such diseases have on the global food production is on the same level than the effects of climate disaster. Currently, global research efforts are aiming for overall estimations on the global scale impacts diseases and pests are going to have on key crops worldwide. For this historical record, predictions for the global climate, simulations and remote sensing are used to calculate risks that have to be expected. These new insights can help to effectively react towards the increasing influences of more extreme weather conditions or crop pathogens. For example, observation data showed a significant linkage between the occurrence of specific pathogens and global warming and presented implications of this linkage for plant breeding and food production.<br>
-				We also asked Wei Xiong for advice for our modelling of fungicide resistance development. He stated, that it is generally possible to create such a mathematic model to calculate these results as long as you have enough mathematic data to establish such a model. There are algorithms to estimate the risk of specific pathogens based on, for example, remote sensing and weather information. After all, resistance to fungicides is more or less an ecosystem process, it depends on a broad range of environmental factors, like the soil, the weather or the type of fungi.<br>
-				He and his research group are still working on modelling the impact and mutation of rust fungi and mainly focused on interactions between the pathogen and its environment. They fed these data into a statistical model they created to estimate the development of rust pathogens. By incorporating said algorithm into a wheat mechanical model effect of rust pathogens could be assessed. They used the NWHEAT model, which was embodied in DSSAT. This model worked well to simulate this development.<br> Moreover, other models like for example Stics could still be introduced.<br>
-				In the end, we also asked Wei Xiong about the most important fungal crop pathogens in China and East Asia. He replied, that the most influential pathogenic fungi to mention here would be a set of rust pathogens, like for example leaf rusts or stem rust.<br>
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-											<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Verena Haberlah-Korr</b><br>
-										Prof. of plant production and protection<br>
-				South Westphalia University of Applied Sciences<br>
-				Soest, Germany</div>
-										<hr style="color:#f1f2f3">
-										Prof. Dr. Verena Haberlah-Korr helped us to comprehend the hazards, fungicides are posing to ecosystems. Her annotations incused our view towards fungicides and their specificity, especially the extent of their hazard in comparison to the unease of the public opinion.
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-											<div class="content conLeft"> <div> First contact 04.10<br>
-				Phone call 16.10<br>
-				We approached Prof. Haberlah-Korr because of her extensive knowledge about plant protection and ecology and wanted to ask her about the impact fungicides have on ecosystems.<br>
-				Prof. Haberlah-Korr pointed out, that the impact of the fungicide depends massively on the kind of fungicide that is used as well as its mode of action. Besides that, fungicides can have negative side effects regardless of if they are biological or chemical reagents. Biological alternatives like copper particles are widely used. These particles can accumulate in the soil but are not harmful for humans in the doses they are used in.<br>
-				A very broad range of fungicides is used with very different modes of action. Some of them can have insecticidal off-target effects or can have negative impacts on soil or water systems.<br>
-				Despite the public opinion, fungicides do not have a massive negative influence if they are used properly. Besides that, their usage can be justified in many cases, since our level of food production would not be feasible without them.<br>
-				Many fungicides work quite specific and do not pose a threat through off-target effects. Others, like many azoles, have a broader mode of action and can even show endocrine effects in mammals. Recently, many fungicides have been revaluated because of stricter regulatory limits, but still for example endocrine effects are difficult to measure.<br>
-				The Federal Office of Consumer Protection and Food Safety monitors the effects of different fungicides and their potential hazards.<br>
-				Meanwhile, farmers often try to avoid fungicides by more carefully choosing their crop strains or are using resistant plants.<br>
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-												<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Professor Peter Langridge</b><br>
-												International Science Coordinator, Wheat Initiative <br>
-												School of Agriculture, Food and Wine – The University of Adelaide <br>
-												Adelaide, Australia</div>
-												<hr>
-												Professor Peter Langridge helped us to coordinate our contact to the Wheat Initiative and forwarded us to experts from all around the world. As the International Science Coordinator at the Wheat Initiative he could also give us great insights into current research projects of interest. Moreover, he pointed out, that we would have to consider that approaches like ours require sophisticated protocols to test the new system before their usage could be allowed in the environment.
-												<div class="content conRight"> <div>
-													First contact 02.08
-				Skype conference 13.08
-				We first came into contact with Prof. Peter Langridge after approaching the Wheat Initiative, a global initiative that establishes, coordinates and strategically organizes research in developing and developed countries on an international level. Moreover, the Wheat Initiative provides a platform for communication between the research community, the funders, policy makers and stake holders. They also support activities and events for this cause.
-				After we got in touch via e-mail, we eventually had a Skype conference and discussed our project, as well as how the Wheat Initiative could support us and who in the Wheat Initiative could help us best.
-				Further, we described our project and our current progress to Prof. Peter Langridge, he highlighted, that the uptake into the fungus resembles a problem in most similar approaches, because fungi are quite complex. Also, he considered our project a good idea, since there is an enormous economic interest in new alternative approaches like our system to fight crop pathogens. Moreover, there is a growing interest in “non-chemical” alternatives that are supposed to lower the negative impact on the ecologic systems. At the Wheat Initiative they are also researching what happens if you would have to farm wheat without using any chemical compounds like pesticides.
-				Furthermore, he pointed out, that the regulations on actually applying systems like our Troygenics in the field can be quite challenging and time-consuming. For example, in the US, there are some regulated and some unregulated forms of CRISPR/Cas and new measures would have to be tested on their classification.
-				Prof. Peter Langridge further pointed out, that the Wheat Initiative is a great platform to get into contact with more experts and also forwarded us to a whole range of expert from all around the world.
-				At the end, he listed some programs and events to reach out to, which might help us gaining attention, like the 1st International Wheat Congress in Sasketchewan, Canada, some workshops about molecular measures in plant breeding at the end of September as well as some Newsletter we could present our project in.
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-													<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Dr. Ravi P. Singh</b><br>
-												Distinguished Scientist - Head-Wheat Improvement Global Wheat Program<br>
-				International Maize and Wheat Improvement Center (CIMMYT)<br>
-												Texcoco, Mexico </div>
-												<hr style="color:#f1f2f3">
-												Dr. Singh gave us information about the possible applicability in rural regions of Africa or Asia and evaluated our approach and its applicability. Moreover, he pointed out some important characteristics of our system that would have to be improved to receive an optimal protection of crops in the real world.	<br>
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-													<div class="content conLeft"> <div> First Contact 05.09<br>
-				We reached out to Dr. Singh as a contact at the International Maize and Wheat Improvement Center (CIMMYT), an international non-profit agricultural research and training organization that is connecting scientists and research programs worldwide to advance in crop protection for the two most important cereal grains in the world: maize and wheat.<br>
-				Dr. Singh pointed out, that the International Maize and Wheat Improvement Center has mainly set its focus on keeping damages through fungal pathogens under control by using host resistances of the plants. Furthermore, fungicides are rarely used by smaller farmers in Asia or Africa. Keeping this in mind, the usage of our Troygenics would be quite complicated to realise in those African and Asian regions, since there is no infrastructure to distribute our system in an easy way. Also, our Troygenics are designed in a way, that they could be applied with the tools that are commonly used by farmers to apply fungicides, but without them being used, distributing the respective tools would be an additional problem.<br>
-				Another point he criticized, was that the targeting and neutralizing of one single fungal pathogen would not avert the threat of fungal damages for the crop. Because of the high variety of fungal pathogens in a country or across countries. To effectively protect the crops a broader applicability would probably be necessary, he concludes. Beyond that, in his opinion this method would have to be implemented into the host plant but like our system, this would be genetic engineering and Dr. Singh did not expect such a measure, based on transgenic organisms, to be applicable in the real world, since the legal situation would clearly prevent such a system from being accessible for farmers.<br>
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-														<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Åsmund Asdal</b><br>
-														Coordinator of Operation and Management <br>
-														Svalbard Global Seed Vault <br>
-														Svalbard, Norway</div>
-														<hr>
-														Åsmund Asdal furthered our understanding of the resilience of fungal plant pathogens under extreme conditions. Which is important information to consider when fighting the spores of fungi on the seed before the planting.
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-															First contact 02.10
-				We came in touch with Åsmund Asdal because of his position at the Svalbard Global Seed Vault in Spitsbergen in one of the northernmost parts of Norway. The Svalbard Global Seed Vault is a secure seed bank for collections of seeds from all around the world. For our project, we wanted to find out if problems with fungi occur even under these extreme conditions.<br>
-				The staff at the Svalbard Global Seed Vault did not have problems with fungi and also did not take any measures to additionally sterilize the stored seeds. The facility is solely for the storage of the seed, nations send in as backups. The staff of the vault is not responsible for the sterilization of the seeds. Besides that, the sent in boxes are never being opened by the staff of the vault.<br>
-				To research the resilience of seed borne fungi disease, the Nordic Gene bank started a seed storage experiment in 1986 in a remote coal mine near Longyearbyen. The objective of this experiment is the monitoring of the longevity of seeds as well as the seed born plant pathogens on the seeds themselves.<br>
-				The experiment was planned for 100 years. After the first 30 years, the first results were published in “Seed longevity and survival of seed borne diseases after 30 years conservation in permafrost - Report from the 100-year storage experiment” in 2019.<br>
-				After 30 years under permafrost conditions, the level of disease on the seeds stayed almost the same. Moreover, all initial seed born plant pathogens have still been observable.<br>
-				The experiment of the Nordic Gene bank convincingly portrays the extreme resilience of fungal pathogens.<br>
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-															<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Teja Tscharntke</b><br>
-														Department of Agroecology<br>
-														University of Göttingen<br>
-														Göttingen, Germany</div>
-														<hr style="color:#f1f2f3">
-														Prof. Dr. Teja Tscharntke gave us his evaluation of potential negative impacts of fungicides on ecosystems. This gave us more information to better highlight some of the advantages of our Troygenics in comparison to commonly used pesticides.
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-															<div class="content conLeft"> <div> First contact 13.08
-														Phone call 15.08<br>
-														We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project.
-														During a phone call, we discussed different parts of our novel approach.<br>
-														Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system.
-														For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize.
-														Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one.  But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany.  In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful.
-														Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products.
-														Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant.
-														Upon discussing the  specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism.
-														Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.
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-																<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Lutz Schmitt</b><br>
-																Chair of Biochemistry I <br>
-																Heinrich Heine University Düsseldorf <br>
-																Düsseldorf, Germany</div>
-																<hr>
-																Prof. Dr. Lutz Schmitt let us acknowledge the social perception of genetic engineering in a better way. Especially, the discrepancy between the contrary security measures and potential mistrust in applications for medicine and agriculture are important points we have to take into consideration for the presentation of our project to people from outside the field.
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-																	First contact at German iGEM Meetup in Düsseldorf 05.07
-				Mail contact 25.07
-				We first came into contact with Prof. Dr. Lutz Schmitt at the iGEM Meetup Germany 2019 in Düsseldorf and contacted him again a few weeks later to refer on his statements.
-				Prof. Schmitt focussed on the discrepancy between the public perception between the usage of genetic engineering in pharmaceutical and agricultural biotechnology. While using these methods for medical purposes is widely accepted by the society, genetical engineering, that is used to improve food production is even considered unethical by parts of the society.
-				Prof. Schmitt reasoned this with the higher willingness of people to take uncommon approaches to maintain their health. In these kind of situations any measures that assured the survival of the patient would be taken into consideration. On the other hand, regarding agriculture, a strict rejection of genetic engineering and the associated methods can be observed in the society. Since the people in Europe are not threatened by shortages of our food supply the necessity of improving the food production is much lower in the first place. Thereby, progression in the use of said methods is being impended by a lack urgency to improve the food production.
-				Besides that, genetic engineering has become a topic that is often used for political elections or similar agendas and has become kind of a bad issue for many people.
-				For our project, this is especially important for the possible implications of our system. Since we are developing our Troygenics as a platform system, that can be adapted to a broad range of pathogens through minor changes of the system itself, we have to be aware of the opportunities it could resemble for medical and agricultural applications but should anticipate the opposition against its use for food production by many people. To avoid this, we should focus even more on explaining our security precautions and have to point out the possibilities our system could represent.
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-																	<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Tessa Alexanian</b><br>
-																iGEM Human Practice Committee<br>
-																iGEM Headquarter<br>
-																Oakland, Canada</div>
-																<hr style="color:#f1f2f3">
-																We had a Skype conference with Tessa Alexanian from the iGEM Human Practice Committee. We described our efforts for Human Practice to her and asked her some questions regarding the topic. She also pointed out why Human Practice is important for iGEM projects and how it can be integrated into our work.
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-																	<div class="content conLeft"> <div> First Contact: 19.10
-				Skype Conference 02.10
-				We approached Tessa Alexanian as a member of the iGEM Human Practice Committee to talk about the value of Human Practice for iGEM and beyond. Moreover, we received a general evaluation of our Human Practice.<br>
-				We talked about the possible risks from an accidental environmental release as well as rights around the world with regards to the release of GMOs, which is something we worked on in our team team before. Afterwards, we talked about different approaches to regulating scientific research and to transfer it to the field. This gave us a great overview about important topics of Human Practice and gave us examples on how Human Practices can be used.<br>
-				Furthermore, we talked about judging in general and the interaction with judges at the Giant Jamboree. Tessa also explained to us, that integrating Human Practices into your project is often a question of how your work as changed by the interactions with experts etc.. Human Practice efforts should fit into your overall story, she concluded.<br>
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-           <a class="anchor" id="h3"></a>
-					<h1 id=a3 style="margin-top:0px; color:#330036"> Project </h1>
-					<hr style="background-color:#330036"> <!--Blaue Linie-->
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-						<img class="roundLeft" src="https://2019.igem.org/wiki/images/8/85/T--Bielefeld-CeBiTec--HP_Schaffrath.jpg">
+						<img class="roundLeft" src="https://2019.igem.org/wiki/images/4/4f/T--Bielefeld-CeBiTec--HP_Dr._Gopaljee_Jha.jpg">
-					<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr.  Ulrich Schaffrath</b><br>
+					<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Gopaljee Jha</b><br>
-Group leader Department of Plant Physiology <br>
+					PhD. student <br>
-RWTH Aachen University <br>
+					National Institute of Plant Genome Research - NIPGR<br>
-Aachen, Germany </div>
+					New Delhi, India</div>
-<hr>
+					<hr>
-Prof. Schaffrath advised us on many aspects of our project. He helped us to overcome some early project ideas, that would have been too difficult to realise in the set amount of time,  advised us on focusing on our Cas System to assure specificity and helped us to decide which wheat strains would fit our proof of concept best and how to access them. He also sent us further information to support our research.  </button>
+					Gopaljee Jha furthered our research and influenced some of our decisions and sparked new ideas on how to induce endocytosis in fungi. Additionally, he gave us some insights into the legal situation of genetic engineering in India.
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-						First contact 25.06 <br>
+						First Contact 05.09
-						Skype conference 23.07 <br>
+	Skype Conference 06.09
+	Gopaljee Jha is doing his PhD. At the National Institute of Plant Genome Research (NIPGR) in New Delhi and focused his Research on plant microbe interactions.<br>
+	After we presented our project and our situation, we discussed several aspects of our work.<br>
+	He suggested us to alternatively think about modifying mycoviruses for our project, which could be a focus of later research.<br>
+	He pointed out, that special bacteria, called Burkholderia, are able to express proteins which can destroy the cell walls of fungi. As far as they were researched, this worked in every fungus that was tested.<br>
+	Additionally, he mentioned that fungi are also capable of quorum sensing, like bacteria. Because of this, he expected hormones to be a better possibility to induce endocytosis. At this point, we already decided to use mating factors for this purpose and have been affirmed by this advice.<br>
+	Gopaljee Jha underlined, that the cell walls of yeasts and fungi differ in many ways and tests with yeast are thereby not representative for the functionality in fungi. This was reflected in our proof of concept, by introducing Aspergilli as filamentous fungi as a second organism to test our system in.<br>
+	In general, Gopaljee Jha assessed specificity through endocytosis to be difficult to achieve, since many fungi are very closely related.<br>
+	As far as he knew, there is no gene that is expressed by all pathogenic fungi during infection but expected that we should be able to find some via RNA analysis if we put enough effort into it.<br>
+	Regarding the legal situation of genetic engineering in India, Gopaljee Jha stated, that because India is located between the USA and the EU its market is influenced by both. As a result, transgenic organisms would be almost impossible to set free under legal conditions. To some extent, specially breed variations of crops should be possible, though. At least as long as they are interspecific.<br>
-						We approached apl. Prof. Dr. Ulrich Schaffrath from the Department of Plant Physiology at RWTH Aachen University to gather further information about pathogenic fungi and their impact on different aspects of society. Besides that, we hoped for an evaluation of our project and some advice for the optimization of our early concepts.
+					</div>
-						We discussed our project and some questions during a Skype conference.<br>
-						The working group of Prof. Schaffrath is doing research on pathogenic fungi that damage crops like cereals, for example wheat, barley and rice. Asian Soybean Rust is of especially high interest because it has the largest economic impact. A part of his research group is also working on the transformation of fungi.
-						Prof. Schaffrath pointed out, that fungicide resistance has been an underrated problem for quite some time now. Meanwhile, resistances have become an even bigger problem and new strategies to fight them are urgently searched for.
-						For example, the plant pathogen stripe rust diminishes the area of chlorophyll of the plant and thereby reduces its yield. Because they need lower temperatures, they only spread around the UK but meanwhile a new strain emerged that also thrives under the warmer climate in countries like Germany.
-						Furthermore, the wheat stem rust strain Ug99 had overcome all resistances implemented into plants that have been used against it and poses a threat to whole harvests in all affected areas.
-						A new counter strategy for the fight against pathogenic fungi, is the usage of a mixture between different reagents and coordinated usage of different fungicides at different times.
-						Research has shown, that only a single nucleotide mutation is required to potentially gain resistance in pathogens against a fungicide. Because of this, resistances can emerge after one or two years of fungicide usage.
-						Because only one base pair has to change to overcome such a pesticide, pathogens like wheat blast cannot be treated effectively with fungicides, which has led to huge problems in Brazil and other parts of the world. These pathogens make the necessity of new approaches even more urgent.
-						Regarding the realisation of our system, Prof. Schaffrath described the legal situation in Germany and the EU as the biggest barrier.
-						For the induced uptake via endocytosis he indicated that finding the right surface ligands will be one of the hardest tasks to accomplish, since many pathogens are not well researched. Moreover, many pathogenic fungi are quite hard to study in the lab. The wheat stem rust (Puccinia graminis f.sp tritici) for example is an obligatory biotrophic organism which cannot be grown in a petri dish. After we focussed on P. graminis at the beginning of our project, we shifted our attention on other pathogens after our discussion with Prof. Schaffrath.<br>
-						While achieving specificity through the surface ligands, can be difficult to achieve for some species, accomplishing specificity through the Cas 13a is more likely to be successful, since many fungal genomes are characterised quite well. We decided to make this part the most important one to assure the specificity of our Troygenics, additional to the other measures like the specific ligands.
-						Another approach we discussed, was the idea to use mycoviruses as alternatives to our Troygenics. Prof. Schaffrath mentioned to not be an expert in this field, but we still managed to evaluate this topic. Since mycoviruses are not transmitted easily between fungi but rather through the fusion of hyphae and asexual reproduction their uptake would be rather difficult to achieve. If the mycoviruses would be sprayed onto the fields like fungicides or the Troygenics, the uptake would be estimated to be rather low in comparison.<br>
-						Another topic we talked about was, if the usage of fungicides or the usage of pathogen resistant plants would be more effective to which he replied, that both are important measures of agriculture and are equally useful and necessary. Resistance genes interacting with pathogen proteins by protein-protein-interaction can be overcome by the pathogen quite easily by point mutations. Therefore, plant breeders began stacking resistance genes to lower the risk of these preventive measures losing their effect. Because plant breeding requires a lot of time, fungicides are often needed as a faster counter measure against new pathogens. However, fungicides might impact the environment and therefore must be used carefully and new, more precise versions of fungicides have to be developed. Furthermore, to lower the negative impact on the environment modern pesticides are applied at very low concentrations to fit the changing legal situation. The optimal solution to this would be perfectly working resistant plants, but for now, fungicides are still required. A well-managed mixture of both measures should be the optimal approach nowadays.
-						We also talked about genetic engineering and its public perception and concluded, that the public discussion is often held on an emotional level. While many scientists are in favour of genetic engineering, it has also become a topic often misleadingly used for politics. The unease about genetic engineering should be evaluated critically, as people often disapprove genetically altered food while openly accepting new medications based on these methods. Moreover, there are products accepted by the society that have been produced using genetically altered organisms, like recombinant proteins, which are used in the production of cheese. However, these proteins do not require to be declared as genetic engineering.
-						To preserve our abundant food supply, genetic engineering would represent an important tool.<br>
-						For our proof of concept, we wanted to know the most common strain of wheat in our area to conduct our experiments under realistic conditions. According to Prof. Schaffrath, there is no such thing as the one most common strain of wheat for Germany. Instead, the optimal strain varies locally and from year to year as well as regarding to the location in comparison to, for example its distance to the coast. He also advised us on contacting a plant breeding companies like KWS to get access to wheat seeds, which we implemented later on during our project.<br>
-						As further important crop pathogens he named Phytophthora infestans, the potato blight, and Asian Soybean Rust but also some bacteria and crop damaging insects. Besides that, he explained that fungi can have a very negative influence on trees as well. There are fungi that can destabilize trees and endanger whole species. Further, trees in cities have to be cut down regularly due to these fungi infections.
-						Regarding pathogenic fungi for humans, he explained the problem is taking a similar course as antibiotic resistant bacteria.  He suspected a higher acceptance of our system as in a medical application opposed to an agricultural tool.
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+							<img class="roundRight" src="https://2019.igem.org/wiki/images/3/3d/T--Bielefeld-CeBiTec--HP_Kogel.jpg" alt="Bild hoffentlich rund">
-						<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Gabi Krczal</b><br>
+						<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Karl-Heinz Kogel</b><br>
-Director of AlPlanta – Institute of plant research<br>
+					Head of the institute for Phytopathology<br>
-Neustadt an der Weinstrasse, Germany</div>
+	University of Giessen<br>
-<hr style="color:#f1f2f3">
+					Giessen, Germany</div>
-Prof. Dr. Gabi Krczal notably furthered our point of view on genetic engineering from a political point of view and gave us new insights into the political processes on an european level. Moreover, she gave us great advice on how to optimize our project to be easily applicable as an alternative to common methods, gave us information about the process of approving a new reagent for agriculture and helped us to get in touch with politicians.
+					<hr style="color:#f1f2f3">
-She also advised us to use reporter genes for the easy verification of the function of our system.
+					We approached Prof. Dr. Karl-Heinz Kogel as an expert of Phytopathology to receive further information about important fungal crop pathogens of economic importance. During a call he advised us on a range of pathogenic fungi and potential concepts of fighting them by implementing latest research into our project.
-</button>
+					</button>
-						<div class="content conLeft"> <div> First contact 13.08
+						<div class="content conLeft"> <div> First contact 25.06<br>
-Phone call 15.08<br>
+	Phone call 19.08<br>
-We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project.
-During a phone call, we discussed different parts of our novel approach.<br>
-Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system.
-For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize.
-Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one.  But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany.  In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful.
-Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products.
-Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant.
-Upon discussing the  specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism.
-Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.
-				</div>
+	We contacted Prof. Dr. Karl-Heinz Kogel, head of the institute for phytopathology at the University of Giessen (Germany), to gather additional information on fungal crop pathogens, their impact and their ecological and economic value.<br>
+	After we explained what the iGEM competition is and our project we discussed the different parts of our work.<br>
+	Prof. Kogel pointed out, that our project has to be planned with a lot of foresight, since the current political and social opinion on genetic engineering would prevent such an application in the near future. With the legal situation regarding this topic in the EU he did not expect a system like our Troygenics to be applicable anytime soon.<br>
+	Besides that, he still liked our approach and considered it a creative idea. Although, he also mentioned, that most comparable approaches do not try to solve this many problems at once, which makes our project susceptible to more problems in direct comparison.<br>
+	He was especially interested in our approach of an induced endocytic uptake into the pathogenic fungi via specific ligands on the surface of the Troygenics.<br>
+	As an additional, similar approach he proposed the idea of using only dsRNA instead of the Troygenics and gene silencing instead of CRISPR/ Cas. While this concept would elute some of the problems of our system, like the required uptake of, in comparison, larger particles	into the target cell, it would also establish new ones. On one hand this would prevent GMOs to form on the fields. This would make it more realistic to implement and is closer to the current praxis. On the other hand, this would require the use of siRNAs which are distinctly more instable and way larger amounts of them would be needed to achieve the same effect. Moreover, this alternative approach would lack the shuttle our concept provides. Finally, the biological production of the Troygenics via E. coli would be a clear advantage in direct comparison.<br>
+	Although, this was a great suggestion, we still considered our approach as a better solution to generate a broader range of potential applications.<br>
+	Furthermore, Prof. Kogel gave us additional information about pathogenic crop fungi and their impact on agriculture. As a further potential target, that would be important to come by, he named Fusarium graminearum as an important pest of wheat and corn plants all around the world. He even mentioned some genes of certain pathways to target for our system. In this case, the pathogen usually is treated with azole-based pesticides, which deactivate proteins essential for the infection of the plant.<br> F. graminearum has become a substantial threat to the food security of the named crops.<br>
+	Regarding Puccinia graminis, a devastating wheat pathogen, that managed to develop multiresistant strains and is threatening the food supply in parts of Africa and that is spreading continuously, he confirmed the danger of the pathogen and added, that its impact is going to increase dramatically because of the progressing climate change.<br>
+	Prof. Kogel also confirmed, that problems due to fungicide resistance are already a relevant threat to our food security and supply. A far range of papers are published about this topic.<br>
+	At the end, we asked Prof. Kogel about methods to distinguish between different spores of fungi and to determine their species without sequencing parts of their genome. Prof. Kogel explained, that the morphology of the spores of different fungal species are quite easy to distinguish with a closer investigation via microscopy.<br>
+</div>
+					</div>
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-								<img class="roundLeft" src="https://2019.igem.org/wiki/images/7/73/T--Bielefeld-CeBiTec--HP_Prof._Dr._Holger_B._Deising2.jpg">
-							<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Holger Deising</b><br>
-						Professor for Phytopathology and Plant Protection<br>
-						Martin-Luther-Universität Halle-Wittenberg<br>
-					Halle, Germany </div>
-						<hr>
-						At the end of August, we got in touch with Prof. Holger Deising at the Marin-Luther-Universität Halle-Wittenberg. Working together with him, we designed a CeDIS specialized for Colletotrichum graminicola, a fungal corn pathogen. He also offered us to test this system in his lab, which we sadly could not do within the short time-frame of iGEM. </button>
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+								<img class="roundLeft" src="https://2019.igem.org/wiki/images/6/61/T--Bielefeld-CeBiTec--HP_Wei-Xiong.jpg">
+							<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Wei Xiong </b><br>
+							Crop Modeler and Physiologist at CIMMYT’s Sustainable Intensification Program <br>
+							International Maize and Wheat Improvement Center – CIMMYT, Chinese Academy of Agricultural Sciences <br>
+							Beijing, P.R. China </div>
+							<hr>
+							Wei Xiong furthered our knowledge about how a mathematic model for the development of fungal resistances can be established and which data sets this would require. He also gave us insights into where these models are used, how important they are and gave us examples how they could be implemented into measures to protect food production.<br>
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-								Phone call: 30.8.19 and 17.09.19<br>
+								First Contact 21.09
+	We reached out to Wei Xiong as a member of the International Maize and Wheat Improvement Center (CIMMYT) in China after we read about his former research, investigating in the influences of weather conditions on harvest in Eastern Europe.<br>
+	We asked him about how changes of the weather and the influence of climate change is estimated to impact the crop production in Europe. While his work only not considered the influence of crop pathogens, but he stated, recent research estimated that the magnitude of the effects such diseases have on the global food production is on the same level than the effects of climate disaster. Currently, global research efforts are aiming for overall estimations on the global scale impacts diseases and pests are going to have on key crops worldwide. For this historical record, predictions for the global climate, simulations and remote sensing are used to calculate risks that have to be expected. These new insights can help to effectively react towards the increasing influences of more extreme weather conditions or crop pathogens. For example, observation data showed a significant linkage between the occurrence of specific pathogens and global warming and presented implications of this linkage for plant breeding and food production.<br>
+	We also asked Wei Xiong for advice for our modelling of fungicide resistance development. He stated, that it is generally possible to create such a mathematic model to calculate these results as long as you have enough mathematic data to establish such a model. There are algorithms to estimate the risk of specific pathogens based on, for example, remote sensing and weather information. After all, resistance to fungicides is more or less an ecosystem process, it depends on a broad range of environmental factors, like the soil, the weather or the type of fungi.<br>
+	He and his research group are still working on modelling the impact and mutation of rust fungi and mainly focused on interactions between the pathogen and its environment. They fed these data into a statistical model they created to estimate the development of rust pathogens. By incorporating said algorithm into a wheat mechanical model effect of rust pathogens could be assessed. They used the NWHEAT model, which was embodied in DSSAT. This model worked well to simulate this development.<br> Moreover, other models like for example Stics could still be introduced.<br>
+	In the end, we also asked Wei Xiong about the most important fungal crop pathogens in China and East Asia. He replied, that the most influential pathogenic fungi to mention here would be a set of rust pathogens, like for example leaf rusts or stem rust.<br>
-We repeatedly talked to Prof. Holger Deising, positioned at the Martin-Luther-Universität Halle-Wittenberg. He advised us to have a look at another fungus: Colletotrichum graminicola. It is a fungus pathogenic for corn and has previously been used to test RNAi systems.<br>
+							</div>
-We were curious, whether he thought our system could work in this fungus and he stated that we could just try it out. Following up on this exciting example we discussed all our subsystems and thought about which one we could test for C. graminicola. Upon learning from Prof. Deising, that endocytic uptake is not an issue for using our system in this fungus, we thought it might be interesting if our CeDIS would work.<br>
-Discussing this possibility, Prof. Deising mentioned that we could come and visit him in his lab to transform the fungus with our CeDIS, actually enabling us to test it in a real-world corn pathogen.<br>
-During our first talk, we discussed the basics of using our system in this fungus: which genes would we want to target, and which promoters should we use to express it.<br>
-Since we wanted to use inducible or repressible promoters to be able to properly estimate the efficiency of our CeDIS, allowing us to distinguish whether the fungus just did not grow or got targeted by it, Prof. Deising suggested using an iron-dependent promotor. He also stated that, for this initial test, the genes would not have to be essential: there were some genes he stated he was sure they would be expressed in the conditions we would grow them in.
-While we did talk to him two more times and discussed our design adapted to C. graminicola, we sadly did not get to visit him in his lab due to the limited time within the iGEM-competition.<br>
-						</div>
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-									<img class="roundRight" src="https://2019.igem.org/wiki/images/6/68/T--Bielefeld-CeBiTec--HP_Cox.jpeg" alt="Bild hoffentlich rund">
+									<img class="roundRight" src="https://2019.igem.org/wiki/images/2/2a/T--Bielefeld-CeBiTec--HPExpert.png" alt="Bild hoffentlich rund">
-								<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Russel Cox</b><br>
+								<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Verena Haberlah-Korr</b><br>
-						Research group leader – Synthetic Biology<br>
+							Prof. of plant production and protection<br>
-						Leibniz University Hannover - Institute of Organic Chemistry<br>
+	South Westphalia University of Applied Sciences<br>
-					Hannover, Germany</div>
+	Soest, Germany</div>
-						<hr style="color:#f1f2f3">
+							<hr style="color:#f1f2f3">
-																	Prof. Dr. Cox furthered our knowledge about fungi, their importance for the industry and ecology and the common methods to work with them. He affirmed our decision to use a Cas system to fight the pathogenic fungi instead of expressing toxic metabolites. Furthermore, he advised us on our lab application and underlined, that the testing of a filamentous fungi aside to the yeast would be very reasonable. </button>
+							Prof. Dr. Verena Haberlah-Korr helped us to comprehend the hazards, fungicides are posing to ecosystems. Her annotations incused our view towards fungicides and their specificity, especially the extent of their hazard in comparison to the unease of the public opinion.
-								<div class="content conLeft"> <div> First contact 19.09
+							</button>
-Video conference 29.07<br>
+								<div class="content conLeft"> <div> First contact 04.10<br>
-Prof. Dr. Russel Cox leads a research group that focusses on the biosynthesis of natural products by fungi using methods of Synthetic Biology. We discussed our project during a Skype conference and evaluated some ideas we had together.<br>
+	Phone call 16.10<br>
-He assured us the importance of projects like ours, since these are essential to face the growing demand for food in the future and to secure the food supply. Besides that, the danger of fungi gaining resistance to fungicides becomes more and more threatening and the impact of fungi on food production or the health care system can be expected to increase drastically in the future due to climate change.<br>
+	We approached Prof. Haberlah-Korr because of her extensive knowledge about plant protection and ecology and wanted to ask her about the impact fungicides have on ecosystems.<br>
-He also affirmed that fungi are of great importance for the Industry of Biotechnology but still have an enormous unused potential. Although they are used on an industrial scale many fungi are still not well established. For this, a new method to accelerate the transformation and selection steps would be an important tool.<br>
+	Prof. Haberlah-Korr pointed out, that the impact of the fungicide depends massively on the kind of fungicide that is used as well as its mode of action. Besides that, fungicides can have negative side effects regardless of if they are biological or chemical reagents. Biological alternatives like copper particles are widely used. These particles can accumulate in the soil but are not harmful for humans in the doses they are used in.<br>
-The importance of fungi for the industry is increasing drastically in recent times. They are being used in fermentation processes on an industrial scale in sectors like medicine and food production, for example for penicillin.<br>
+	A very broad range of fungicides is used with very different modes of action. Some of them can have insecticidal off-target effects or can have negative impacts on soil or water systems.<br>
-Since he and his research group are investigating in metabolites, we discussed the usage of toxic metabolites to fight pathogenic fungi. Prof. Dr. Cox estimated, that 4-5 different complex genes would have to be expressed to efficiently work for fungi. Through our discussion he affirmed our plan to use a Cas System for our system, since it would be easier to apply for fungi.<br>
+	Despite the public opinion, fungicides do not have a massive negative influence if they are used properly. Besides that, their usage can be justified in many cases, since our level of food production would not be feasible without them.<br>
-Regarding our lab application, Prof. Dr. Cox mentioned, that most substances can be produced with fungi, although this often requires a huge amount of work. The problem of these processes rather concerns the slow growth rate of fungi, the difficult transformation and the specific integration. Because of this CRISPR is often regarded as the easier choice. Also, research with CRISPR as transformation method for fungi is being conducted. An important goal for improving the work with fungi would be a faster method for transformation.<br>
+	Many fungicides work quite specific and do not pose a threat through off-target effects. Others, like many azoles, have a broader mode of action and can even show endocrine effects in mammals. Recently, many fungicides have been revaluated because of stricter regulatory limits, but still for example endocrine effects are difficult to measure.<br>
-For our proof of concept, he advised us on thinking about yeast and filamentous fungi in different ways because filamentous fungi are considerably more complex. Filamentous fungi are also becoming increasingly important for industrial processes.<br>
+	The Federal Office of Consumer Protection and Food Safety monitors the effects of different fungicides and their potential hazards.<br>
-Prof. Dr. Cox especially underlined the fact that our system has to be really specific. A system that would target fungi in general would be disastrous for the ecosystem.<br>
+	Meanwhile, farmers often try to avoid fungicides by more carefully choosing their crop strains or are using resistant plants.<br>
 </div>
+							</div>
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-									</div>
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-										<img class="roundLeft" src="https://2019.igem.org/wiki/images/0/0e/T--Bielefeld-CeBiTec--HP_Dr._Primrose_J_Boynton.jpg">
+										<img class="roundLeft" src="https://2019.igem.org/wiki/images/7/71/T--Bielefeld-CeBiTec--HP_Langridge.jpeg">
-									<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Primrose Boynton</b><br>
+									<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Professor Peter Langridge</b><br>
-									Project Leader<br>
+									International Science Coordinator, Wheat Initiative <br>
-									Max Planck Institute for Evolutionary Biology - Max Planck Fellow Group Environmental Genomics<br>
+									School of Agriculture, Food and Wine – The University of Adelaide <br>
-								Plön, Germany</div>
+									Adelaide, Australia</div>
 									<hr>
-									Dr. Primrose Boynton repeatedly supported us by giving us advice on working with yeast. She helped us to optimize our method of cultivation and gave us access to one of the strains. Her advice on how to avoid flocculation in yeast during cultivation helped us to integrate the received strains into our project and compare them with related yeast strains. Furthermore, her help enabled us to analyze more cultures of Saccharomyces cerevisiae with our microfluidic chip.
+									Professor Peter Langridge helped us to coordinate our contact to the Wheat Initiative and forwarded us to experts from all around the world. As the International Science Coordinator at the Wheat Initiative he could also give us great insights into current research projects of interest. Moreover, he pointed out, that we would have to consider that approaches like ours require sophisticated protocols to test the new system before their usage could be allowed in the environment.
+									<div class="content conRight"> <div>
+										First contact 02.08
+	Skype conference 13.08
+	We first came into contact with Prof. Peter Langridge after approaching the Wheat Initiative, a global initiative that establishes, coordinates and strategically organizes research in developing and developed countries on an international level. Moreover, the Wheat Initiative provides a platform for communication between the research community, the funders, policy makers and stake holders. They also support activities and events for this cause.
+	After we got in touch via e-mail, we eventually had a Skype conference and discussed our project, as well as how the Wheat Initiative could support us and who in the Wheat Initiative could help us best.
+	Further, we described our project and our current progress to Prof. Peter Langridge, he highlighted, that the uptake into the fungus resembles a problem in most similar approaches, because fungi are quite complex. Also, he considered our project a good idea, since there is an enormous economic interest in new alternative approaches like our system to fight crop pathogens. Moreover, there is a growing interest in “non-chemical” alternatives that are supposed to lower the negative impact on the ecologic systems. At the Wheat Initiative they are also researching what happens if you would have to farm wheat without using any chemical compounds like pesticides.
+	Furthermore, he pointed out, that the regulations on actually applying systems like our Troygenics in the field can be quite challenging and time-consuming. For example, in the US, there are some regulated and some unregulated forms of CRISPR/Cas and new measures would have to be tested on their classification.
+	Prof. Peter Langridge further pointed out, that the Wheat Initiative is a great platform to get into contact with more experts and also forwarded us to a whole range of expert from all around the world.
+	At the end, he listed some programs and events to reach out to, which might help us gaining attention, like the 1st International Wheat Congress in Sasketchewan, Canada, some workshops about molecular measures in plant breeding at the end of September as well as some Newsletter we could present our project in.
-<div class="content conRight"> <div>
-										First contact 16.08<br>
-										Dr. Primrose Boynton is conducting research on Saccharomyces yeasts and their interactions with their environment at the Max Planck Institute for Evolutionary Biology in Plön (Northern Germany).<br>
-										We approached her to receive information about yeast strains and their phylogenetic relationship for our proof of concept. Over the course of our project she advised us on several aspects of our work. To demonstrate the specificity of our Troygenics, we came up with some experiments to compare the reaction of Troygenics targeted on Saccharomyces cerevisiae on said strain of yeast and its closest relative Saccharomyces paradoxus.<br>
-										Based on her given information we wanted to use the Saccharomyces paradoxus strains 5696 and N44. Besides giving us access to the strains, Dr. Boynton advised us on how to best work in the lab with those strains, for example, by sharing her experience on the cultivation media.<br>
-										At a later point of our project, she advised us on how to avoiding flocculation of the yeast strains during cultivation. Since the flocculation of yeast cultures massively complicates the determination of the optical density it can resemble a problem. Beyond that, the flocculation of yeast stopped us from cultivating them in our microfluidic chips. Dr. Boynton pointed out, that the level of Calcium in the media is known to be the most important factor for the tendency of yeasts to form flocs. Furthermore, she recommended to ad small amounts of EDTA to the cultivation media to prevent this effect. When yeast cultures are floculating, the comparison and quantification of the growth and thereby the influence of our Troygenics would be almost impossible. <br>
-										Additionally, floculating cultures can hardly be analyzed with microfluidic devices.<br>
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@@ Line 889: / Line 647: @@
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-											<img class="roundRight" src="https://2019.igem.org/wiki/images/b/bb/T--Bielefeld-CeBiTec--HP_Mark_Varrelmann.jpg" alt="Bild hoffentlich rund">
+											<img class="roundRight" src="https://2019.igem.org/wiki/images/f/f0/T--Bielefeld-CeBiTec--HP_Dr._Ravi_P._Singh.jpg" alt="Bild hoffentlich rund">
-										<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Mark Varrelmann</b><br>
+										<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Dr. Ravi P. Singh</b><br>
-									Research group leader - Phytomedicin<br>
+									Distinguished Scientist - Head-Wheat Improvement Global Wheat Program<br>
-								University of Göttingen – Institute for sugar beet research (IfZ)<br>
+	International Maize and Wheat Improvement Center (CIMMYT)<br>
-							Göttingen, Germany</div>
+									Texcoco, Mexico </div>
 									<hr style="color:#f1f2f3">
-									Dr. Mark Varrelmann furthered our understanding of the impact pathogenic fungi have on agriculture and highlighted some aspects we must focus on for our project to be applicable for this problem. He also established contact between us and other experts in this field. Most importantly, he advised us to extend our proof of concept by the filamentous fungi Aspergillus niger to better resemble a filamentous crop fungus.  </button>
+									Dr. Singh gave us information about the possible applicability in rural regions of Africa or Asia and evaluated our approach and its applicability. Moreover, he pointed out some important characteristics of our system that would have to be improved to receive an optimal protection of crops in the real world.	<br>
-</button>
+									</button>
-										<div class="content conLeft"> <div> Phone call 21.06<br>
+										<div class="content conLeft"> <div> First Contact 05.09<br>
-										We had an extensive phone call with Prof. Dr. Mark Varrelmann from the Institute for sugar beet research (IfZ) at the University of Göttingen. As the Research group leader for Phytomedicine he was able to give us a lot of insights into the impact crop damaging fungi have on our agriculture and food production in general. He also gave us an overview about the current situation of research for these kinds of fungi and the latest ideas and methods to fight these pathogens.<br>
+	We reached out to Dr. Singh as a contact at the International Maize and Wheat Improvement Center (CIMMYT), an international non-profit agricultural research and training organization that is connecting scientists and research programs worldwide to advance in crop protection for the two most important cereal grains in the world: maize and wheat.<br>
-										We discussed our early concept of our project and asked him to evaluate its functionality. According to him our project is feasible even as a completely novel approach, but we have to take some bottlenecks into consideration. All in all, he estimates our project to be very sophisticated and challenging.<br>
+	Dr. Singh pointed out, that the International Maize and Wheat Improvement Center has mainly set its focus on keeping damages through fungal pathogens under control by using host resistances of the plants. Furthermore, fungicides are rarely used by smaller farmers in Asia or Africa. Keeping this in mind, the usage of our Troygenics would be quite complicated to realise in those African and Asian regions, since there is no infrastructure to distribute our system in an easy way. Also, our Troygenics are designed in a way, that they could be applied with the tools that are commonly used by farmers to apply fungicides, but without them being used, distributing the respective tools would be an additional problem.<br>
-										The most important problems have advised us to focus on were the Endocytosis into the fungus, the surface ligands for the uptake of our Troygenics and the adaptability to a wider range of fungi, since fungi are an extremely diverse group of organisms.<br>
+	Another point he criticized, was that the targeting and neutralizing of one single fungal pathogen would not avert the threat of fungal damages for the crop. Because of the high variety of fungal pathogens in a country or across countries. To effectively protect the crops a broader applicability would probably be necessary, he concludes. Beyond that, in his opinion this method would have to be implemented into the host plant but like our system, this would be genetic engineering and Dr. Singh did not expect such a measure, based on transgenic organisms, to be applicable in the real world, since the legal situation would clearly prevent such a system from being accessible for farmers.<br>
-										The Endocytosis is problematic because the cell walls of fungi are considerably more complex than cell membranes of bacteria and resemble a quite selective barrier between the fungus and its surroundings. Often there is little research conducted on the cell walls of certain species of fungi. Dr. Varrelmann considered the endocytosis uptake of larger particles, like our Troygenics as an especially challenging task to achieve.<br>
+</div>
-										Furthermore, he estimated, that the specific surface ligands our system is using to initiate specific uptake into the targeted crop pathogen are limited by the current level of research on this field.<br>
+									</div>
-										Often the molecular composition as well as the function of some fungal cell wall structures is lesser understood than it would be necessary for our project. So, to realise our project for a larger range of targets, we would have to put some thoughts into how phytopathogenic surface proteins could be identified, since they have not been characterized yet. He highlighted, that a thorough knowledge about surface proteins would be necessary to realise our system as an application for a special fungus. He also added, that genome databases often only contain a small amount of information about phytopathogens.<br>
-										Most importantly, Dr. Varrelmann advised us to extend our proof of concept with yeast by an additional filamentous fungus to reduce the difference to a, mostly filamentous, pathogenic fungi. Therefore, he suggested Aspergillus niger for this addition, as it represents a well-studied member of the filamentous fungi and has a broad application in the biotechnology industry. Since we are aiming to create a new lab application based on our system to transform fungi and the potential targeting of pathogenic fungi for humans, like some Aspergilli species, a model organism closer to a pathogenic fungus is crucial for out project. Therefore, we decided to integrate Dr. Varrelmanns suggestion into our project.<br>
-										Last, but not least, Dr. Varrelmann introduced us to several different fungal crop pathogens of economic importance and lead us to further research about this. Moreover, he put us into contact with other molecular mycologists that helped us to optimise our project.<br>
-											</div>
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-												<img class="roundLeft" src="https://2019.igem.org/wiki/images/1/11/T--Bielefeld-CeBiTec--HP_Dr._Olaf_Kniemeyer.jpg">
+												<img class="roundLeft" src="https://2019.igem.org/wiki/images/2/2a/T--Bielefeld-CeBiTec--HPExpert.png">
-											<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Olaf Kniemeyer</b><br>
+											<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Åsmund Asdal</b><br>
-											Molecular and Applied Microbiology · Deputy head of Department<br>
+											Coordinator of Operation and Management <br>
-											Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI)<br>
+											Svalbard Global Seed Vault <br>
-										Jena, Germany </div>
+											Svalbard, Norway</div>
 											<hr>
-											We got in touch with Dr. Olaf Kniemeyer to gather further information about Aspergillus niger and Aspergillus nidulans to optimize our proof of concept. Moreover, we were interested in suggestions of human-pathogenic fungi our Troygenics could possibly target. Last but not least, we received access to a strain of Aspergillus nidulans through Dr. Kniemeyer.<br>
+											Åsmund Asdal furthered our understanding of the resilience of fungal plant pathogens under extreme conditions. Which is important information to consider when fighting the spores of fungi on the seed before the planting.
 											<div class="content conRight"> <div>
-												First Contact 30.08<br>
+												First contact 02.10
-Phone call 02.09<br>
+	We came in touch with Åsmund Asdal because of his position at the Svalbard Global Seed Vault in Spitsbergen in one of the northernmost parts of Norway. The Svalbard Global Seed Vault is a secure seed bank for collections of seeds from all around the world. For our project, we wanted to find out if problems with fungi occur even under these extreme conditions.<br>
-We contacted Dr. Olaf Kniemeyer in the framework of our research regarding Aspergillus niger and Aspergillus nidulans. Moreover, we hoped to receive access to a strain of A. nidulans for our project, since we planned to use this strain to verify the specificity of our Troygenics with our proof of concept.<br>
+	The staff at the Svalbard Global Seed Vault did not have problems with fungi and also did not take any measures to additionally sterilize the stored seeds. The facility is solely for the storage of the seed, nations send in as backups. The staff of the vault is not responsible for the sterilization of the seeds. Besides that, the sent in boxes are never being opened by the staff of the vault.<br>
-After we depicted the iGEM competition, our project and the situation we were in we discussed potential methods to induce endocytosis in Aspergilli.<br>
+	To research the resilience of seed borne fungi disease, the Nordic Gene bank started a seed storage experiment in 1986 in a remote coal mine near Longyearbyen. The objective of this experiment is the monitoring of the longevity of seeds as well as the seed born plant pathogens on the seeds themselves.<br>
-Dr. Olaf Kniemeyer explained, that A. niger is mostly used for applications in biotechnological processes, while A. nidulans is rather used to conduct research on the genetics of Aspergilli.<br>
+	The experiment was planned for 100 years. After the first 30 years, the first results were published in “Seed longevity and survival of seed borne diseases after 30 years conservation in permafrost - Report from the 100-year storage experiment” in 2019.<br>
-As a method to generate an uptake through the fungal cell wall he mentioned the possible usage of amphotericin B which enables an endosomal uptake. For our project this would not be applicable though, because as a polyene it intercalates with the cell membrane to induce said uptake by opening small holes in the cell membrane. By doing so it would also damage liver and kidney tissue, which would make it difficult to apply for therapeutically uses.
+	After 30 years under permafrost conditions, the level of disease on the seeds stayed almost the same. Moreover, all initial seed born plant pathogens have still been observable.<br>
-Since, Aspergilli are able to cause aspergilloses some of the have human-pathogenic characteristics. Around 75% of aspergillosis are caused by Aspergillus fumigatus, but A. niger can also be found responsible for this disease in less common cases. Industrial production strains are considered as bsl 1, like A. nidulans.<br>
+	The experiment of the Nordic Gene bank convincingly portrays the extreme resilience of fungal pathogens.<br>
-Regarding A. nidulans, we asked him for access to a wild type strain and thought of the different steps that have to be taken to receive access to these trains. We later received a lab strain of Aspergillus nidulans per post.<br>
-Dr. Kniemeyer depicted, that common methods to work with these fungi in the lab are fully functional. If we would want to establish our Troygenic as a lab application, the referring method would have to be at least faster, cheaper or more efficient that these commonly used methods.<br>
-As further important human-pathogenic fungi, that could be targeted with our system, Dr. Kniemeyer named especially A. fumingatus and some other fungi. In the USA he named some Cryptococcus strains. These fungi would resemble important possible targets our Troygenic could be constructed for.<br>
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-													<img class="roundRight" src="https://2019.igem.org/wiki/images/2/26/T--Bielefeld-CeBiTec--HP_Annicka_Roos.jpg" alt="Bild hoffentlich rund">
+													<img class="roundRight" src="https://2019.igem.org/wiki/images/1/14/T--Bielefeld-CeBiTec--HP_Prof._Dr._Teja_Tscharntke.jpg" alt="Bild hoffentlich rund">
-												<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Annika Roos</b><br>
+												<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Teja Tscharntke</b><br>
-											Marketing consultant for wheat (Nordrhein-Westfalen)<br>
+											Department of Agroecology<br>
-											KWS Lochow GmbH<br>
+											University of Göttingen<br>
-										Bergen, Germany</div>
+											Göttingen, Germany</div>
 											<hr style="color:#f1f2f3">
-																					Mrs. Roos helped us to understand the process of decision which kind of wheat fits best under certain circumstances, helped us to decide which kind of wheat strain we would need for our proof-of-concept and ultimately got us access to the strains for using them in our project. </button>
+											Prof. Dr. Teja Tscharntke gave us his evaluation of potential negative impacts of fungicides on ecosystems. This gave us more information to better highlight some of the advantages of our Troygenics in comparison to commonly used pesticides.
-												<div class="content conLeft"> <div> First contact 07.08<br>
+											</button>
-Multiple contacts via e-mail and phone for organizational purposes<br>
+												<div class="content conLeft"> <div> First contact 13.08
-We reached out to Mrs. Annika Roos who is the marketing consultant for wheats in our part of the country at KWS Lochow GmbH, a leading distributor of seeds for agriculture in Germany and beyond.<br>
+											Phone call 15.08<br>
-For our proof-of-concept, we were planning to cultivate wheat under lab conditions to conduct some tests on the plants. To recreate conditions as close to the regional agriculture as possible, we wanted to find out which strain of wheat is the most common and resilient, so we could use it for our project.<br>
+											We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project.
-Annika Roos taught us, that there is no such thing as the the most used wheat strain in Germany. Due to differing weather conditions, soil composition or general demands on wheat strains, the choice of the optimal wheat strain has to be taken by every farmer, each year independently and often relies on complex consultancy to optimize said choice.<br>
+											During a phone call, we discussed different parts of our novel approach.<br>
-After this clarification, Mrs. Roos helped us to make a decision on which wheat strains would fit our needs best and which factors and characteristics have to be taken into consideration for this decision. One of these factors would be whether we should use pickeled or un-pickeled seeds.<br>
+											Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system.
-Ultimately, Mrs. Roos got us access to the strains we decided to use. We received three different strains, each one stained and un-stained. We got the strains “KWS Talent”, “KWS Emerick” and “KWS Fontas” who differ in the quality of the harvest, the yield and their susceptibility to weather or potential fungicides respectively.<br>
+											For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize.
+											Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one.  But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany.  In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful.
+											Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products.
+											Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant.
+											Upon discussing the  specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism.
+											Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.
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+													<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Lutz Schmitt</b><br>
-																					<img class="roundLeft" src="https://2019.igem.org/wiki/images/a/a6/T--Bielefeld-CeBiTec--HP_Fischer.jpeg">
+													Chair of Biochemistry I <br>
-																				<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Reinhard Fischer</b><br>
+													Heinrich Heine University Düsseldorf <br>
-													Professor at the Institute for Applied Biosciences, Department of Microbiology <br>
+													Düsseldorf, Germany</div>
-													Karlsruhe Institute of Technology – KIT <br>
-													Karlsruhe, Germany </div>
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-													We contacted Professor Reinhard Fischer as an expert for Aspergillus. He furthered our understanding of the processes we had to manipulate to induce endocytosis. Besides that, he affirmed our proceeding for our proof of concept and supported our research to realize an uptake of our Troygenics by the targeted fungi.
+													Prof. Dr. Lutz Schmitt let us acknowledge the social perception of genetic engineering in a better way. Especially, the discrepancy between the contrary security measures and potential mistrust in applications for medicine and agriculture are important points we have to take into consideration for the presentation of our project to people from outside the field.
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+													<div class="content conRight"> <div>
-																					First contact 16.08<br>
+														First contact at German iGEM Meetup in Düsseldorf 05.07
-Skype conference 04.09.<br>
+	Mail contact 25.07
-We contacted Professor Fischer as an expert for the molecular biology of Aspergillus and other fungi and asked him for an evaluation of our proof of concept. During a Skype conference we received a chance to discuss different aspects of our project.<br>
+	We first came into contact with Prof. Dr. Lutz Schmitt at the iGEM Meetup Germany 2019 in Düsseldorf and contacted him again a few weeks later to refer on his statements.
-Like we heard before, he pointed out, that the legal situation especially in Germany and the EU would make it really difficult to make a system like the Troygenics commercially usable<br>
+	Prof. Schmitt focussed on the discrepancy between the public perception between the usage of genetic engineering in pharmaceutical and agricultural biotechnology. While using these methods for medical purposes is widely accepted by the society, genetical engineering, that is used to improve food production is even considered unethical by parts of the society.
-Regarding our proof of concept, he affirmed that inducing endocytosis in Aspergillus niger or Aspergillus nidulans would assure the applicability in the respective other fungus. Since we based our proof of concept partly on A. niger, this was an important information for the further testing of our system, because the genetics of A. nidulans is better understood than the one of A. niger. Therefore, including research on A. nidulans has made it easier for us to find a method to induce endocytosis in A. niger.<br>
+	Prof. Schmitt reasoned this with the higher willingness of people to take uncommon approaches to maintain their health. In these kind of situations any measures that assured the survival of the patient would be taken into consideration. On the other hand, regarding agriculture, a strict rejection of genetic engineering and the associated methods can be observed in the society. Since the people in Europe are not threatened by shortages of our food supply the necessity of improving the food production is much lower in the first place. Thereby, progression in the use of said methods is being impended by a lack urgency to improve the food production.
-We further discussed our current status in our approach. Professor Fischer suggested to test the possibility of inducing endocytosis using peptide transporters of the cell. We implemented this in our endocytosis-design for A. niger. We utilized a proline transporter as a target to enable endocytosis of our Troygenics.<br>
+	Besides that, genetic engineering has become a topic that is often used for political elections or similar agendas and has become kind of a bad issue for many people.
-He also acknowledged our approach of targeting essential genes of the fungi to prevent the bypassing of our system through mutation as well as our approach of blasting the gRNAs we use to target the fungus, making sure that they do not appear in any other organisms. By doing so, we disabled our Cas-system from accidentally damaging other organisms and causing unwanted off-target effects. Professor Fischer also named us some potential target genes.<br>
+	For our project, this is especially important for the possible implications of our system. Since we are developing our Troygenics as a platform system, that can be adapted to a broad range of pathogens through minor changes of the system itself, we have to be aware of the opportunities it could resemble for medical and agricultural applications but should anticipate the opposition against its use for food production by many people. To avoid this, we should focus even more on explaining our security precautions and have to point out the possibilities our system could represent.
-He also mentioned the general current discussion regarding genetic engineering in fungi and the respective legal situation. He also mentioned different seminars and discussions in the near future that cover this topic.
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-																					<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Richard Oliver</b><br>
-																						Professor of Agriculture<br>
-<br>
-													Centre for Crop Disease Management (CCDM) at Curtin University<br>
-												Curtin, Australia</div>
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+														<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Tessa Alexanian</b><br>
+													iGEM Human Practice Committee<br>
+													iGEM Headquarter<br>
+													Oakland, Canada</div>
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-													Prof. Richard Oliver helped us to improve our project in various ways. He advised us on how to induce endocytosis reliably and how to design our CeDIS in a way to minimalize the chance of pathogens overcoming it by mutation. This was a major influence on our modeling project. Beyond that, we worked together to optimize our concept of effective appliance of our system and the verification of the functionality our system. Last but not least, he helped us to improve the purification process of the Troygenics from the E. coli cultivation broth to optimize their applicability. </button>
+													We had a Skype conference with Tessa Alexanian from the iGEM Human Practice Committee. We described our efforts for Human Practice to her and asked her some questions regarding the topic. She also pointed out why Human Practice is important for iGEM projects and how it can be integrated into our work.
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-																						First contact 14.08
+														<div class="content conLeft"> <div> First Contact: 19.10
-Skype conference 15.08<br>
+	Skype Conference 02.10
-We first got in touch with Prof. Richard Oliver through the forwarding of Professor Peter Langridge of the Wheat Initiative. As Professor of Agriculture at the CCDM, the Centre for Crop Disease Management at Curtin University (Australia), Prof. Richard Oliver had accumulated some knowledge we could use to optimize our project.<br>
+	We approached Tessa Alexanian as a member of the iGEM Human Practice Committee to talk about the value of Human Practice for iGEM and beyond. Moreover, we received a general evaluation of our Human Practice.<br>
-Regarding the bottleneck of endocytosis that many experts had mentioned before, he pointed out that researchers had thought about using endocytic uptake in fungi before and that there had been a focus in research on this since.<br>
+	We talked about the possible risks from an accidental environmental release as well as rights around the world with regards to the release of GMOs, which is something we worked on in our team team before. Afterwards, we talked about different approaches to regulating scientific research and to transfer it to the field. This gave us a great overview about important topics of Human Practice and gave us examples on how Human Practices can be used.<br>
+	Furthermore, we talked about judging in general and the interaction with judges at the Giant Jamboree. Tessa also explained to us, that integrating Human Practices into your project is often a question of how your work as changed by the interactions with experts etc.. Human Practice efforts should fit into your overall story, she concluded.<br>
-He considered our approach as feasible, since fungi can take up larger molecules from their surroundings. Binding one of these molecules to our Troygenic could thereby, theoretically, induce its endocytic uptake. For comparison, Prof. Richard Oliver mentioned the cholera toxin, which, in a similar manner, consists out of two parts that induce the uptake by the cell and respectively its toxicity.
+													</div>
-We investigated the alternative CeDIS idea of introducing siRNAs into our system, but after discussing it we discarded the idea. We did not expect this approach to work in all species of fungi and moreover, this would not allow us to create a lab application, that we are aiming for with our Troygenics.
+															</div>
-Regarding other aspects of our CeDIS, Prof. Richard Oliver advised us to increase the number of genetical targets to prevent the targeted fungi from circumventing our Troygenics through mutation. He suggested to use three different targets to assure the applicability of our system. As a result, of this recommendation we also focused our modeling on the question of the optimal amount of gRNAs for our system.
+</div>
-Moreover, Prof. Richard Oliver gave us insights into the combination of methods like fungicides, analogous reagents and plant breeding to protect harvests. He advised us to come up with a similar concept, to assure the integrated functionality of our approach. To proceed with this idea, he also named us a broad range of further experts and associations to get in contact with.
+</div>
-As a potential formulation he suggested using clay nanoparticles that could be attached to our Troygenics and applied to the field. He pointed out that, since they are cost-effective and widely applicable materials, they could be of great use for our project. Contrary to this, we discussed emulating the usage and formulation of currently used fungicides to keep the amount of necessary adaptation to apply our system minimal. However, designing a clay nanoparticle-based system to achieve a slow release system of the Troygenics could open whole new possibilities and potential new applications for our project.<br>
-Another addition he made addressed our proof of concept and the verification of the functionality of our system. To investigate the effects our Troygenics would have on plants, we bred wheat plants ourselves to apply our system on them in a controlled environment. In that case we should be able to evaluate the effects of our applied Troygenics based on the visible changes of the plants. For this experiment Prof. Richard Oliver advised us to use specially altered strains of Arabidopsis thaliana with fluorescent reporter genes for the detection of physical damage to the plant. Using these special strains would improve the reliability and the quantification of the potential damage to the plant and would be more precise than an examination of regular wheat plants by eye. He also gave us some lists where we should be able to find said strains and experts to contact to gain access to them.<br>
-Prof. Richard Oliver further advised us to watch out for microscopic defense responses of the plant after our tests. If these should occur the composition of the mixture containing our Troygenic probably has to be reconsidered.<br>
-A further optimization we designed with Prof. Richard Oliver and implemented into our work was the purification process for the application on the plants. Since our Troygenics are produced in E. coli, just concentrating the Troygenics after discarding cells and debris and using the concentrated supernatant would be an obvious approach. What we did not take into consideration up to that point was that many cultivation media like LB are containing components from bacteria that could cause a PAMP (Pathogen-associated molecular pattern) mediated immune reaction in the plant. Because of this, applying a direct concentration of the supernatant would probably trigger immune reactions of the plant and would thereby become useless for our application. This was one of the reasons why we decided to purify our Troygenics prior to continue working with them. We also thought about that when improving the purification protocol.
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-																													<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Susanne Zeilinger-Migsich</b><br>
-																						Deputy Head of the Department of Microbiology <br>
-																						University of Innsbruck <br>
-																						Innsbruck, Austria </div>
-																						<hr>
-																						Prof. Dr. Susanne Zeilinger-Migsich evaluated our concept of inducing endocytosis in our targeted fungi and gave us some great advice. She specifically suggested to have a closer look on mating pheromones to induce the specific uptake of our Troygenics. We implemented this for the proof-of-concept in S. cerevisiae cells.<br>
-Furthermore, she helped us to get in touch with other important experts
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-																														First contact 29.07<br>
-Phone call 06.08<br>
-We approached Prof. Dr. Susanne Zeilinger-Migsich as a Professor for Microbiology and asked her to review some aspects of our project. During a phone call she gave us advice on endocytosis and our lab application.<br>
-As a mycologist, she pointed out that fungi have a cell wall that would be a particular obstacle to overcome. Also, the structure and composition of the cell wall can differ from fungus to fungus. Yet, she thought an endocytotic uptake like it was planned for our project would be possible. Depending on the structure and state of the cell wall certain substances can also be adsorbed but would not be taken up into the cell.<br>
-She mentioned that filamentous fungi can communicate via pheromones. By doing this, male and female fungi are able to recognize each other, while asexual fungi can be more complicated. The receptors for such signals are really sensitive and only tiny amounts of the receptor-bound substance are taken up via endocytosis. A substance like this could be sufficient to generate an uptake of our Troygenics, Prof. Zeilinger-Migsich estimated. After this, we focused on researching these kinds of molecules.<br>
-For S. cerevisiae we used the Mating factor alpha as a promising pheromone that does induce endocytosis. We lateron showed experimentally that the uptake initiated by it is specific for S. cerevisiae. <br>
-We also asked for further information that might help to estimate how helpful our system might be for lab work. We wanted to know more about fungal metabolites that are hard to produce, and whether our system could maybe present an improvement for the productivity of these. Prof. Dr. Zeilinger-Migisch explained, that genetic engineering or specialized signaling would be necessary to achieve the production of many substances, because most gene clusters for secondary metabolites are turned off in most fungi used in lab applications. On the other hand, substances that are being produced on a commercial scale are produced with already existing high-performance strains, specialized on products like penicillin or cephalosporin. So, the most relevant future achievements would be finding new interesting metabolites by switching on silent gene clusters.<br>
-At the end, she additionally helped us to get in touch with experts on yeast and the medical view on fungi from Innsbruck (Austria) as well as with experts for Aspergillus at the Institute of Technology in Karlsruhe (Germany).<br>
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-																														<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Robert Park</b><br>
-																															Judith and David Coffey Chair of Sustainable Agriculture and
-							Director of Cereal Rust Research at the University of Sydney Plant Breeding Institute
-<br>
-University of Sydney<br>
-																						Sydney, Australia</div>
-																						<hr style="color:#f1f2f3">
-																						Prof. Robert Park shared his extensive experience regarding rust fungi with us and affirmed the idea of using mating factors to induce endocytosis in yeast. Moreover, he helped us to decide to use Aspergillus for our proof of concept to characterize our system. Furthermore, he gave us great insights into different models regarding pathogenic fungi over the course of the last decades and pointed out potential struggles with modeling their gaining of resistances.
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-																														<div class="content conLeft"> <div> First contact 13.08
-																															First contact 20.08
-								Skype conference 26.08<br>
-								We came into contact with Prof. Robert Park after a forwarding of one of the other experts of the wheat initiative we discussed our project with. Prof. Park is conducting research about cereal rust and sustainable agriculture.<br>
-								Regarding our project, we discussed different aspects of our work as well as the problems we had encountered. As we considered the endocytosis as the most important bottleneck of our project, he suggested to use mating proteins to initiate endocytosis. Since we had been using them for yeasts, he stated that these could not only work for those, but that they also exist in rust, even though they do not use sexual recombination. There stil would be further research necessary to determine if this would a valid approach. He also suggested further literature to read about this. Since there is only little published on rust, like he pointed out himself, this has been very helpful.<br>
-								He also named a set of further potential pathogens for our system to target. For example, the pathogens Ustilago and Tilletia that resemble important pathogens for maize and wheat. These two are also known for the research on their mating but, as pathogens, must be handled according to higher biosafety levels. Despite the fact that they do usually live as parasites, these fungi can be grown in artificial media. Furthermore, he suggested some additional fungi for the testing of our system, that rather resembled the crop pathogens we wanted to fight. Since they were rather difficult to obtain, we have not been able to get access to these strains for our project int this short amount of time.<br>
-								Prof. Robert Park also gave us general information about his research on rust pathogens and explained, that Mildew or Septoria are having comebacks lately because of their insensitivities to many commonly used reagents.<br>
-								Together, we discussed the suitability of different fungi for our proof of concept but ultimately decided to stick with Aspergillus for the characterization and later adapt the system to fungi, closer related to the actual crop-damaging fungi. This discussion also settled our choice of using Aspergillus and not adding further fungi with a closer resemblance of actual crop pathogens, mostly due to time constraints. <br>
-								As an addition, Prof. Park pointed out, that some fungi are dikaryotic and getting the Cas into both nuclei would probably decrease the effectiveness of the system, since our Cas do not has to enter the nucleus, this would not apply to our system.<br>
-								For our modeling Prof. Robert Park evaluated the parameters we planned to take into consideration and underlined, that the actual rate of mutations in rust fungi is really hard to estimate, since the number of random mutations can vary significantly and depends on a lot of different factors. Moreover, many essential genes would be unsuitable for a targeting due to the rapid evolution of rust fungi, he stated. He also gave us an overview about different approaches of models, that have been developed regarding pathogenic fungi.<br>
-								For our project, he highlighted the importance of the specificity to assure the biosafety of our application. It is really important to reassure people, that the new application does not damage things unintentionally. The public perception is extremely important for these kinds of approaches.<br>
-								At the end, Prof. Park underlined once more, that while more research has to be conducted on rust fungi prior, projects like ours are going to have an important impact in the future since the knowledge about these pathogens increases constantly. He pointed out, that our system cold really have a positive impact. We just need some further research.<br>
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-																															<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Eduardo Antonio Espeso Fernández</b><br>
-																														Research Professor at the Dept. of Cellular and Molecular Biology <br>
-																														Centro de Investigaciones Biológicas (C.S.I.C.) <br>
-																														Madrid, Spain </div>
-																														<hr>
-																														Prof. Espeso Fernández supported the progress of our research by helping us to comprehend the biosafety levels of Aspergillus niger and Aspergillus nidulans and furthered our knowledge about endocytotic uptake in fungi. After his suggestions, we discarded saccharides as potential ligands for our Troygenics.
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-																																First contact 22.08<br>
-We approached Prof. Eduardo Antonio Espeso Fernández to gather further information about methods to induce endocytosis in Aspergillus and to receive an experienced opinion on the biosafety level of Aspergilli.<br>
-Prof. Espeso Fernández is working with Aspergillus nidulans, which has been used as a model organism for decades. He was interested in our project, because of its therapeutic application in fungi.<br>
-We asked him for methods of transformation for Aspergillus nidulans and Aspergillus niger and differences between them. Prof. Espeso Fernández stated, that both fungi can be transformed, although his expertise rather concerns A. nidulans.<br>
-Since we were using A. niger as a part of our proof of concept, although A, nidulans is better understood genetically, we asked about the biosafety levels about the two fungi and if it would be sensible to also use A. nidulans or research A. nidulans for methods appliable for both fungi. While A. niger is considered GRAS (generally accepted as safe) A. nidulans is not, but there are no special needs to handle the strain, Prof. Espeso Fernández pointed out. Both fungi have been used as a model in the past but for the use in biotechnological processes, A. niger would be preferable. He also considered A. niger a reasonable choice, since we wanted to use a GRAS organism for our project.<br>
-Prof. Espeso Fernández displayed, that an uptake of larger particles would maybe be hard to achieve, because Aspergilli usually secretes enzymes to search for nutrients. Carbon sources and amino acids are taken up through more or less specific transporters in the cell wall. Disaccharides and Polysaccharides are processed in the extracellular medium to be taken up afterwards. As a result, using a certain sugar or similar nutrient as a ligand to generate a specific uptake is not expected to work. Prof. Espeso Fernández explained that endocytosis is not used for the uptake of nutrients but for the recycling of transporters and signalling machinery of the plasma membrane. A “recognizable cargo” like a nitrogen-source compound could be taken up via induced endocytosis. An example for a similar process would be the urea transporter of some fungi. If this transporter is localized in the plasma membrane and urea as a nitrogen source is depleted in the surrounding medium, the transporter would be initialized as soon as a rich nitrogen source, for example ammonium, is added to the medium. In that case the urea or amino acid-particles could be internalized via endocytosis.<br>
-These comments on our project helped us to progress in our research and to discard saccharides as potential ligands for our Troygenics to induce endocytosis.<br>
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-																																<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. George Diallinas</b><br>
-																														Professor of Biology<br>
-																														National and Kapodistrian University of Athens - Department of Biology - Aspergillus Genetics Lab<br>
-																														Athen, Greek</div>
-																														<hr style="color:#f1f2f3">
-																														We approached Prof. George Diallinas as a Professor for transport proteins in Aspergillus to ask him some questions regarding our proof of concept concerning methods to induce endocytosis in Aspergilli. He gave us the valuable hint to use Aspergillus-specific amino acid transporters to induce endocytosis, like many viruses do.
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-																																<div class="content conLeft"> <div> First contact 22.08<br>
-Skype conference 27. 08<br>
-Mail with helpful papers attached: 30.8.<br>
-In August, we started extensive research on receptor specific endocytosis in Aspergillus niger. Prof. Reinhard Fisher forwarded us to Prof. George Diallinas. At the end of August we had a very informative skype conference. Prof. Diallinas enlightened us about the different Aspergillus-strains and recommended that we should proof our concept in related strains like Aspergillus niduland. He stated, that principles, that work in nidulans will work in niger, too. A disadvantage of niger Diallinas pointed out was, that this strain lacks a sexual cycle so the usage of mating pheromones, that worked properly for S. cerevisiae, was no possible option. <br>
-Together with Prof. Diallinas we worked out it would be a promising option to use a virus-like approach. He told us that many viruses use cell-specific transporters to get actively internalized by the target cell. Although he stated that he has little expertise on mycoviruses, he suggested us to target PrnB, an aspergillus-specific proline transporter, with a short proline-peptide fused to our Troygenics.<br>
-On the 30th of August, Prof. Diallinas forwarded us some very useful and interesting papers on viruses and how they exploit cellular transporters to get inside their target cell which helped us to design an endocytosis-inducing fusion-protein for A. niger.<br>
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-																																	<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Lara Petersen</b><br>
-																																	Uni Bielefeld – BCIII <br>
-																																	Bielefeld University</div>
-																																	<hr>
-																																	Lara Petersen helped us in many ways regarding the optimal handling procedures for S. cerevisiae and providing us with different yeast strains and established protocols for transformation and fluorescence microscopy.
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-																																		First contact: June<br>
-Advise on fluorescence microscopy: August<br>
-Supporting performed fluorescence microscopy experiments during September<br>
-As soon as we decided to work with S. cerevisiae as a model organism we approached Lara Petersen to get some helpful advise regarding S. cerevisiae. During a first meeting in June we explained our project to her and received some advise regarding our experimental design for S. cerevisiae. Lateron in August we got in touch with Lara again to talk about the planned fluorescence microscopy experiments.<br>
-As soon as the neccessary preparations were done, we received an introduction into the usage of the provided fluorescence microscope. During September Lara supported us by giving helpful tips to improve the performed fluorescence microscopy experiments and being constantly reachable in the case of further questions.
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-																																		<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Matthias Hahn</b><br>
-																																	Professor of Phytopathology<br>
-																																	University of Kaiserslautern<br>
-																																	Kaiserslautern, Germany</div>
-																																	<hr style="color:#f1f2f3">
-																																	As a plant pathologist, Prof. Hahn could give us great insights into the metabolic workings of fungi and the interaction between plant and pathogen. We discussed and pondered different methods to induce endocytosis and received advice to verify said methods, for example how to implement reporter genes into our concept.<br>
-He also forwarded us to further experts and supported our research with a range of papers.<br>
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-																																		<div class="content conLeft"> <div> First contact 13.08
-																																			First contact 25.06
-Skype conference 19.08<br>
-We established contact to Prof. Dr. Matthias Hahn to receive an evaluation of our project and to gather further information on plant pathogen interaction. Prof. Hahn send us some interesting papers and even parts of his lectures. Later, we also had a Skype conference to ask more complex questions. <br>
-As a phytopathologist, we mainly asked him about the uptake of the Troygenics by the pathogenic fungi. In his opinion, the specific, endocytotic uptake resembled the most critical part of the project, because the cell walls of fungi are quite hard to overcome.<br>
-While our concept was considered a good idea, we also discussed other approaches. For example, we discussed methods using mycoviruses for transformation through the fusion of hyphen or the interaction between the plant and the fungus through RNAi and exosomes. Though these methods show some benefits they did not featured the same rate of uptake or specificity as our concept.<br>
-Overall, he highlighted the need of novel approaches to fight such pathogens and the increasing demand of the industry for them.<br>
-We also talked about early ideas for our modelling with Prof. Hahn. In his opinion, predicting the chance of resistances forming is a really difficult thing to do because for natural mutations to occur only one nucleotide has to be altered and every mutation can be dependent on context for itself. Furthermore, we agreed upon the difficulty of testing our hypothesis empirical in the short timeframe of iGEM.<br>
-At last, he gave us extended information on metabolic functions of fungi and advised us on implementing a reporter gene into our system, since a validation of our methods via fluorescence would be most convenient to verify.<br>
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-																																			<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Alexander Lichius</b><br>
-																																			Department of Microbiology <br>
-																																			University of Innsbruck<br>
-																																			Innsbruck, Austria</div>
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-																																			Dr. Alexander Lichius helped us to affirm some of our decisions for our concept and forwarded us to experts of his local research community.
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-																																				First contact 01.08<br>
-Phone call 02.08<br>
-We came into contact with Dr. Alexander Lichius after reaching out to experts on the Mycology Tyrol Homepage, an interactive online platform for the local mycology research in Tyrol. During a phone call we had a chance to describe our project, our current status, plans and problems. He helped us to get in touch with further experts and advised us on general ideas.<br>
-Dr. Alexander Lichius also stated, that a system that works in Aspergillus nidulans should also work in other Aspergilli, like Aspergillus niger, although he forwarded us to his colleges to receive conformation for this. This information was important for our research, since the genetics of A. nidulans are way better understood than the ones of A. niger, that we used for our proof of concept, since it was easier to obtain and better understood regarding methods for its cultivation.<br>
-He also affirmed our choice of A. niger as an addition for our proof of concept, because of its degree of relationship to other Aspergilli that have proven to be pathogenic for humans.<br>
-Additionally, Dr. Lichius named us a list of important, potentially human-pathogenic fungi, that we could target or use as examples for our outlook.<br>
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-																																				<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Fabio Gsaller PhD.</b><br>
-																																			Institute of Molecular Biology<br>
-																																			Medical University of Innsbruck<br>
-																																			Innsbruck, Austria</div>
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-																																			Dr. Fabio Gsaller helped us to improve and adapt our concept for Aspergillus niger. His evaluations and suggestions improved our planning of the according experiments and his experience with transformations of fungi helped us to plan our project accordingly. Additionally, he forwarded us to further important experts and provided us with information we integrated in our project.<br>
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-																																				<div class="content conLeft"> <div>First contact 02.08<br>
-Phone call 06.08<br>
-We got forwarded to Fabio Gsaller PhD. by Dr. Alexander Lichius. Both work at the Institute of Molecular Biology at the Medical University of Innsbruck (Austria). Dr. Gsaller has long-standing experience in working with Aspergilli and could advise us on the genetic manipulation of Aspergillus.<br>
-During a phone call, we discussed our plans for our project. Although, Dr. Gsaller did not use Cas13a, he had worked with CRISPR/ Cas9 and successfully used the system in Aspergillus fumigatus. While transformation for fungi are quite hard to achieve, it is posible and the protocols for the methods already exist.<br>
-He helped us by providing important information on the procedures to transform A. niger. The fungi have to grow for three or four days after the transformation until the colonies become visible. After this, the colonies have to be transferred to new plates multiple times to be sure that they were actually single colonies and overcame the selection marker. Because of this, a transformation process requires two to four weeks, an amount of time that has to be taken into consideration – especially in a competition like iGEM.<br>
-As Dr. Gsaller had experience with the transformation of Aspergillus fumigatus and Aspergillus nidulans, he offered to check out our protocols for Aspergillus niger if we would like him to. <br>
-He further advised us which lab strain to choose best to and named some scientists we should get into contact with to receive further expertise.<br>
-When we discussed our project, he stated that the largest bottleneck within our concept might be endocytosis. Fungi have a cell wall that can be hard to overcome and many therapeutically approaches fail on this task. However, Dr. Gsaller still assumed that the endocytic uptake might work in A. niger if it also worked in closely related strains like A. nidulans.<br>
-He thought that our aim should be to create a system where only the gRNAs had to be integrated to adapt the system to a fungus, sincea cas system on its own does not seem to have an impact on Aspergilli. <br>
-Moreover, he has sent us further information about A. niger and other fungi that are pathogenic for humans and informed us about their impact. This was important information for evaluating the possible applications of our Troygenics to fight pathogens for humans.<br>
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-					<h1 id=a4 style="margin-top:0px; color:#721356"> CeDIS </h1>
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-        <button class="collapsible colRight"> Max Mustermann (Muster University, Musterhausen) <br>
-                          Head of Muster departement <br>
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-          <button class="collapsible colLeft"> Max Mustermann (Muster University, Musterhausen) <br>
-                            Head of Muster departement <br>
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-            <button class="collapsible colRight"> Max Mustermann (Muster University, Musterhausen) <br>
-                              Head of Muster departement <br>
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-              <button class="collapsible colLeft"> Max Mustermann (Muster University, Musterhausen) <br>
-                                Head of Muster departement <br>
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-					<h1 id=a5 style="margin-top:0px; color:#0b5091"> Lab Application </h1>
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-      <button class="collapsible colRight"> Max Mustermann (Muster University, Musterhausen) <br>
-                        Head of Muster departement <br>
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-        <button class="collapsible colLeft"> Max Mustermann (Muster University, Musterhausen) <br>
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-          <button class="collapsible colRight"> Max Mustermann (Muster University, Musterhausen) <br>
-                            Head of Muster departement <br>
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-            <button class="collapsible colLeft"> Max Mustermann (Muster University, Musterhausen) <br>
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Difference between revisions of "Team:Bielefeld-CeBiTec/Human Practices"

Revision as of 02:59, 22 October 2019

Forward farm

Dr Patrick Beuters

Karl Eschenbacher

Bernd Olligs