Summary
Human Practices are the aspects of iGEM projects that open up the remarkable achievements of the iGEM teams all around the world to the public and reward them with new insights and ideas to their projects.
To develop our Troygenics as a completely novel approach on the transformation of eukaryotic pathogens, a lot of bottlenecks had to be overcome.
We approached experts from numerous fields of research to implement their interdisciplinary suggestions into our project. Integrating their advice and contrary points of view made a huge difference for the realization of our project.
Since our main application was aimed for fighting crop damaging fungi, we got into contact with experts from various fields including agriculture, industry, politics, biosafety, legislation and a large number of researchers.
Additionally, reaching out to farmers, visiting them on their farms, having discussions and optimising the application of our Troygenics accordingly added new facets to our iGEM project and made it more applicable for the real world in the long run.
Human Practices have been one of the major driving forces throughout our project and implementing their suggestions into our work has significantly leaped our project forward multiple times.
First contact 25.06
Skype conference 23.07
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.
We discussed our project and some questions during a Skype conference.
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.
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.
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.
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.
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.
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.
Phone call: 30.8.19 and 17.09.19
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.
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.
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.
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.
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.
First contact 19.09
Video conference 29.07
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.
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.
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.
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.
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.
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.
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.
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.
Phone call 21.06
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.
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.
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.
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.
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.
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.
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.
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.
First contact 07.08
Multiple contacts via e-mail and phone for organizational purposes
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.
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.
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.
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.
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.
Phage Assembly
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Endocytosis
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
CeDIS
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lab Application
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
