H U M A N P R A C T I C E S

Report on Genetic Engineering

Genetic engineering has been a hotly debated topic in politics as well as society in the past decades and still is today. Arguments like the nutrition of a growing world population due to a declining infant mortality rate or the loss of considerable areas of arable land due to erosion or pollution damage keep fueling the controversy whether genetically modified organisms (GMO), especially crops, are needed to sustain the global demand for food. On the opposite, concerns have been raised concerning the potential adverse effects on human health and environmental safety. Besides the facts, part of the public dispute is based around ethical questions and trust issues towards institutions and authorities. There have been studies and surveys carried out addressing many of these topics. Additionally a diverse cluster of organisations and the media is bombarding the public with contrary statements. This report tries to give an overview on mankind's relation towards modifying genetics, a brief summary of used methods, and gathers statements from scientists and authorities. It is meant as the motivational basis for this years Marburg iGEM team´s Public Engagement and Human Practice efforts.

G M O
R E P O R T

Gathering reviews, opinions and statements, this report is meant as the foundation of our Human Practice efforts.

History of Genetic Modification
Our ancestors had no conception of genetics but still were able to influence the genes of multiple organisms. It is a process known to everybody called artificial selection or selective breeding. Those individuals with the most desirable traits, like the biggest and most delicious fruits or the highest loyalty, is chosen to propagate and produce offspring. This process is repeated over several generations and the result is an organism with the selected traits. The dog, existing today in many variations, is believed to be the organism our ancestors selectively bred first around 32,000 years ago (Zimmer, 2013). And there are many more instances like corn which originates from a grass called teosinte with very few kernels (‘Evolution of Corn’, n.d.). However, this process is not considered GMO technology today. What we understand under genetic modification today can be traced back to the mid 1900´s, when scientists discovered that genetic material can be transferred between different species (Avery, MacLeod, & McCarty, 1944), the structure of genetic material was identified as a double helix (Crick, Watson, & Bragg, 1954), the genetic code was deciphered (Nirenberg, Matthaei, Jones, Martin, & Barondes, 1963) and finally a DNA recombinant technology was described (Cohen, Chang, Boyer, & Helling, 1973). Only a few decades after these ground-breaking discoveries were made, the first genetically modified (GM) plants were produced in 1983, which were antibiotic resistant tobacco and petunia (Bevan & Chilton, 1982; Fraley, 1983; Herrera‐Estrella et al., 1983). Soon, the first GM plants were commercialized: In the early 1990´s China approved modified tobacco and in 1994 the United States Food and Drug Administration (U.S. FDA) approved the “FLAVR SAVR” tomato which was modified to have a longer shelf live by delaying ripening. Today, numerous GM plants exist and are in use, covering popular fruits like papaya, melon and apple, flowers like roses, feed plants like sugar beet, vegetables like tomato, maize and potato and even cotton for clothes production (‘GM Crops List—GM Approval Database | ISAAA.org’, n.d.).

Current Numbers on GM Crops
World
As stated above, many GM crops are relevant for food production today, be it indirectly for animal feed in production lines or directly as consumables. In 2018, 26 countries planted 191.7 million hectares worldwide with GM crops, which is an increase of 1% from 2017´s worldwide planted area. Accordingly, since its first commercialization in 1996 with 1.7 million hectares planted, GM crop area increased by an approximate 113-fold. The accumulated area planted with GM crops from 1996 to 2018 was 2.5 billion hectares. This makes biotechnology the fastest adopted crop technology in the world. Of the 193 member nations of the United Nations Organisation (UNO) 42 nations plus the European Union (EU) adopted GM crops, of which 26 countries (21 developing and 5 industrial) planted and 44 imported GM crops. The four major GM crops, namely soybeans, maize, cotton and canola, occupied 99% of the GM crop area (Figure 1). GM crop share in total crop area was 78% for soybeans, 76% for cotton, 30% for maize and 29% for canola. 42% of the global GM crop area was planted with stacked trait crops tolerant to various herbicides and pesticides. Around the world the GM crop area was unevenly distributed with the top five countries United States of America (USA), Brazil, Argentina, Canada and India planting 91% of the global GM crop area. In the EU, the two nations Spain and Portugal planted the GM crop MON810, which is an insecticide resistant maize, together covering 120.990 hectares. 95% of the area was planted by Spain. From 2017 to 2018 GM crop area in the EU has decreased by 8% from 131.535 hectares (Figure 2). Nevertheless the EU imported GM crops, roughly 30 million tons of soybean products, 10 million tons of maize and 2.5 million tons of canola originating from Argentina, Brazil and the USA. Since 1992, across the world 4.349 approvals to GM crops have been issued, of this being 2.063 for food, 1.461 for animal feed use and 825 for cultivation (‘ISAAA Brief 54-2018: Executive Summary | ISAAA.org’, n.d.).

GM crops 2018 — Fig.1 - Area and adoption rate of GM crops (biotech crops) in 2018 worldwide. Adopted from ISAAA, 2018.
*GM sugar beets, potatoes, apple, squash, papaya and brinjal/eggplant.

Fig.2 - Global map of GM (biotech) countries in 2018. Adopted from ISAAA, 2018.

Germany
In Germany, there is no more GM crop farming since 2012. GM maize has been planted last in 2008 (3.171 hectares, 0.15% of total maize area in Germany) and GM potatoes have been planted last in 2011 (2 hectares, 0.0008% of total potato area in Germany). GM crop area never made up more than 0.02% of land used by agriculture in Germany (‘Gentechnik’, n.d.).

Modern Methods in Breeding
The traditional way of breeding, as explained above, although having generated many domestic plants and animals, is relatively slow and limited by the available traits individuals express. Modern breeding methods enhance the trait spectrum and the pace in which new traits can be discovered and implemented to crops and animals.

Plant Mutagenesis
As it is known that practical breeding depends on genetic variation plant mutagenesis expands the variability of traits. Variations found in nature do not represent the original spectra of spontaneous mutations due to the fact that they are recombining within populations and interacting with environmental factors. In the process of mutagenesis heritable changes occur in the genetic information induced by mutagenic agents called mutagens. These mutagens can be of chemical, for instance substances interacting with the DNA, or of physical origin, such as ionizing radiation (Oladosu et al., 2016). After using the mutagen on the crops, mostly seeds, seedlings or cell cultures from which single cells can be grown out, screening has to be done to see if changes in traits have been achieved by mutations. These mutations can be DNA double strand breaks, single base exchanges or alkylation of bases. In most cases, generated mutants are heterozygous, because the mutation happened in only one allele. Therefore the breeder needs to rear subsequent generations to evaluate recessive mutations. Selection then takes place in form of phenotypical, physical or molecular testing to determine for instance plant height, earliness of maturity and biochemical composition. Mutagenesis breeding has impacted agriculture massively with more than 3.300 entries to the Mutant Variety Database (‘Mutant Variety Database’, n.d.), covering all major food and feed crops.

Genetic Engineering
This term is used to describe methods which alter the genetic makeup of an organism using DNA recombinant technology. This technology resorts to enzymatic tools called restriction enzymes. These cut the DNA site specific and can thereby isolate genetic constructs coding for desirable traits. When gene(s) are introduced into an organism this can be achieved either directly or indirectly. The direct approach utilizes a method called microparticle bombardment (Sanford, 1990). Developed in the 1980´s, engineered DNA is coated on microparticles of either gold or tungsten and then shot with high velocity at the target organism using high pressure helium gas. The DNA fragments can then be incorporated into the organism’s genetic material. There are other direct methods such as electroporation or microinjection but particle bombardment is the most effective. The indirect approach makes use of a vector: the soil bacterium Agrobacterium tumefaciens naturally infects plants and alters its hosts genome via a plasmid called Ti-plasmid. This plasmid can be engineered to carry genes coding for a desired trait instead of its natural genes for infection. With the development of a method called CRISPR/Cas9 and other variants genetic engineering in plants got much easier (Cong et al., 2013; DeMayo & Spencer, 2014; Ran et al., 2013). This system is found in bacteria where it serves as a defence mechanism against viruses. The endonuclease is guided to its target cutting site via a guide RNA where it induces a double strand break (DBS). The DBS can be repaired in two distinct ways. Non-homologous end joining leads to a small deletion while homologous recombination allows for the integration of donor DNA into the endogenous DNA. Thereby, the CRISPR method allows for small alteration or whole gene insertions at target sites.
At this point it may be appropriate to introduce the two terms “cisgenic” and “transgenic”. While “transgenic” refers to organisms in which genetic material outside the species boundary, originating from a donor organism which is sexually incompatible to the engineered organism, has been inserted. “Cisgenic” on the contrary describes genetic modifications within the boundaries of sexual compatibility. Therefore, cisgenic plants are similar to traditionally bred plants (Schouten, Krens, & Jacobsen, 2006). The most obvious example of transgenic plants are the many varieties of so called “Bt” crops. Standing for Bacillus thuringiensis, into these plants a gene from the bacterium was integrated which leads to the production of a crystal protein that is toxic to specific pest insects (‘Insecticidal Plants’, 2015).

Opinions on GMOs
There are many scientific publications evaluating specific GMO traits towards the environment and health safety. Additionally many reviews exist summarizing GMO effects to a much broader scale possible here (Bawa & Anilakumar, 2013; Nicolia, Manzo, Veronesi, & Rosellini, 2014; Snell et al., 2012; Zhang, Wohlhueter, & Zhang, 2016). In many of these, authors conclude that the application of GMOs offers great opportunities but still has to be carried out with precautions. A simple “yes” or “no” cannot be given (Zhang et al., 2016). Still, due to the partly contradictory evidence, it cannot be said there is a consensus among scientists, according to Hilbeck et al., 2015.

Benefits of GM Crops
Humanity faces several challenges in the coming decades. Amongst them are the increasing world population, a decrease of arable land or the bottleneck of traditional breeding methods (Zhang et al., 2016). To all of these, GMOs pose a genuine answer. The easiest way to produce more food for a growing population is to increase productivity by earlier maturity, easier harvesting, processing and cultivation. Adding to that, if we resorted to organically producing todays yields, humanity would need to cultivate an additionally 3 billion hectares, which is the equivalent to the size of two South Americas (‘Time to call out the anti-GMO conspiracy theory – Mark Lynas’, n.d.). But food also needs to become more nutritious. A good example here is “Golden Rice” (Ye et al., 2000), which produces a precursor of vitamin A. The deficiency of vitamin A is estimated to kill more than half a million children under the age of 5 each year (Black et al., 2008) and cause another half a million irreversible cases of childhood blindness (Humphrey, West, & Sommer, 1992).

Risks of GM Crops
GMOs pose risks to its consumer as do crops deriving from traditional breeding. Major risks are toxicity, allergenicity and genetic hazards emerging from the inserted or altered gene itself, the expressed protein, products of the metabolism, pleiotropic effects or the disruption of natural genes in the organism (Zhang et al., 2016). There have been reports on the strong allergenicity of “Starlink” maize, which is directly connected to the inserted gene from Bacillus thuringiensis (Bravo, Gill, & Soberón, 2007; Sanchis, 2011; Tabashnik, 1994; Werth, Boucher, Thornby, Walker, & Charles, 2013). Also, GM crops can have an adverse ecological influence. For example, the weed species Amaranthus palmeri did evolve a glyphosate resistance after years of glyphosate use on resistant cotton fields (Gilbert, 2013). Another possibility is the fact, that insect resistant crops infer with ecological food webs by shifting predator prey ratios. Moreover, targeted pests might decline and primary minor pests become major issues (Bawa & Anilakumar, 2013; Snow & Palma, 1997).

Statements from Authorities
The "Public Acceptance of Agricultural Biotechnologies (PABE) project" revealed a range of questions concerning rather institutional considerations of the public, such as who is benifitting from GMO use, by whom consequences have been evaluated, if authorities have enough power to regulate large companies and why the public has not been better informed about GMO usage (Marris, 2001). For this reason, an overview of institutional statements might be appropriate.
The European Commision (EC) published the book “A decade of EU-funded GMO research”. Within this endeavor more than 200 million Euro of research grants were spent to evaluate GMO´s in areas such as environmental impact, food safety, biomaterials, biofuels, risk assessment and management. It conclusively states: “The main conclusion to be drawn from the efforts of more than 130 research projects, covering a period of more than 25 years of research, and involving more than 500 independent research groups, is that biotechnology, and in particular GMOs, are not per se more risky than e.g. conventional plant breeding technologies.” (Publications Office of the European Union, 2010).
The National Academy of Sciences founded by the U.S. Congress summarizes in their comprehensive report, that large numbers of animal feeding studies provided reasonable evidence that animals were not harmed by food derived from GM crops, although admitting some studies were not designed optimal. Furthermore, long-term data in livestock health before and after GM crop introduction did not show adverse effects associated with the crops. And at last, epidemiological data on cancer and human health over time was revised but no substantiated evidence was found that GM crops are less safe than foods from non-GM crops (Read "Genetically Engineered Crops, n.d.).
The British Royal Society states the following to the question “Is it safe to eat GM crops?” on its website: “Yes. There is no evidence that a crop is dangerous to eat just because it is GM. There could be risks associated with the specific new gene introduced, which is why each crop with a new characteristic introduced by GM is subject to close scrutiny. Since the first widespread commercialisation of GM produce 18 years ago there has been no evidence of ill effects linked to the consumption of any approved GM crop.” Before new GM foods are permitted to the market a variety of test has to be completed and the results are used by the authorities to determine the safety of the GM product, making “new GM crop varieties at least as safe to eat as new non GM varieties, which are not tested in this way.” (‘Is it safe to eat GM crops?’, n.d.).

Conclusion
As biologists, using genetic engineering methods every single day, they are quite natural to us. Nevertheless, we are confronted with the public debate too. Having experienced the public aversion towards GMO ourselves and having read about the many proposed justifications against it we realized that a direct exchange between the public and experts from all fields as well as diverse interest groups might provide a good common ground for an open discussion.

D R . N I N A
S C H E E R

Policy makers are caught between public concerns and expert opinions. Communication is key.

P L A N T M A R K E T

Reaching out to senior people, we advertised our panel discussion and adressed public concerns towards genetically modified organims.

Extracting pepper — Fig.1 - Thomas Södler extracting DNA from pepper at the plant breeder festival.

We advertised our panel discussion by running a booth at the plant breeders festival in Marburg on the weekend of 14th September. It is an event for young and old people alike and the perfect platform to bring our discussion closer to a diverse audience.

When we talked with Nina Scheer, member of the German parliament from the Social Democratic Party, she told us that a large part of the population has a very critical attitude towards genetically modified food. We are convinced that the main reason for this critical attitude is a lack of information and communication between scientists and the public that leads to insecurities in the population. In order to get a picture of the public opinion on genetic engineering, we asked the participants to fill out a questionnaire, which we then statistically evaluated. We realized that in particular middle-aged and older people have a critical view on genetic engineering or tend to say from themselves that they don't know enough about it and would like to hear more. Through personal discussions we were able to get in contact with people, raise awareness on the issue and also learned a lot ourselves about the landscape of opinions.

At the same time we gave children and interested adults the chance to take a closer look at our work by letting them experimentally extract DNA from pepper (figure 1). We used this to also explain to the children what DNA is, what it means to them and what exactly they were doing in each step.

Due to the contact and discussion with people from the general society and especially outside of our university background, the plant breeder festival expanded our knowledge and we therefore see it as a great success. We received a lot of positive feedback and spoke personally with many supporters and critics of genetic engineering and took away some lively discussions. In addition, we were able to evaluate around 200 questionnaires and further promote our panel discussion. This was demonstrated by the run on our subsequent panel discussion, where we were happy to recognize some familiar faces from the festival.

Evaluation
In our questionnaire, we first had the participants assess their current state of knowledge about genetic engineering and divided the questions into gender, age and basic attitude.

As far as gender is of interest, most of the participants rated themselves in the criteria as "rather bad" and "medium". Men consider themselves to be better informed than women. Especially in the category "very good" it is mainly men, while in the category "very bad" it is mostly women, regarding their self-assessment. It is possible that men are more self-confident in this aspect and thus, consider themselves to be better informed.

sex — Fig.2 - Answers analyzed by gender for the question "How knowledgable are you about gene editing?".

Fig.3 - Answers analyzed by age for the question "How knowledgable are you about gene editing?".

knowledge — Fig.4 - Answers analyzed by attitude towards gene editing.

In the category “age”, the average self-assessment is also "rather bad" and "medium". However, younger people (18-29) generally considered themselves to be better informed than older people. A problem is that the older generation may have more difficulty accessing information.

It is also noticeable that the participants have a positive attitude towards genetic engineering when they are better informed. This shows that there is not enough information on genetic engineering and that it does not reach the population.

The first part of our study reveals two problems: bad accessibility of information to the older generation creates a discrepancy between the level of knowledge of younger and older people. As a result, older people feel less informed, which in turn leads to skepticism towards genetic engineering. For example, one can focus the source of information on television/radio or newspaper and thus try to reach older people more effectively.There is also a problem with the flow of information in general, as people are obviously better attuned to genetic engineering when they know more about it. In general, there is a need for better information on the subject and more discussions at a political level.

We then asked whether the participants considered genetic engineering in plants, animals and humans to be ethically justifiable.

Most people had a positive attitude towards plants. Here, many people probably recognized the potential, also with regard to the 2050 food problem or resistance to pests.

Surprisingly, many participants are more likely to agree with genetic engineering in humans than in animals. Most people probably think of the potential of genetic engineering to fight serious diseases and thus cure diseases where traditional treatment is not sufficient. In addition, when it comes to genetic engineering on animals, some people may think of animal experiments, which many consider to be reprehensible. In addition, it is potentially possible to ask people before such interventions, while animals have no voice. Our results confirmed us conducting our panel discussion to create another source of information and to inspire as many people as possible to think about it. We hope to be able to educate and hope that the potential of genetic engineering will be recognized by the general public.

sex — Fig.5 - Attitude towards gene editing in plants.

sex — Fig.6 - Attitude towards gene editing in animals.

sex — Fig.7 - Attitude towards gene editing in humans.

P A N E L D I S C U S S I O N

The panel discussion allowed regional residents to debate with experts from various fields about green genetic engineering.

Genetic engineering has been a hotly debated topic in politics as well as in society in the past decades and still is today. New methods in Genome Editing (GE) are making the regulation of methods more and more important due to the increased difficulty to identify changes in the genetic material of manipulated organisms. The European Court of Justice (ECJ) ruling on GE of July 2018 pleased the interest groups of genetic engineering sceptics, but disappointed the research community to a large extent. The panel discussion organised by the iGEM team Marburg 2019 with participants from plant breeding research, industry, ethics research and genetic engineering critics should give the public an insight into the different breeding methods and the according viewpoints of different stakeholders. After a keynote lecture by biochemist Prof. Dr. Lars-Oliver Essen from the Philipps- University of Marburg the panel discussion started. It was moderated by Michael Lange of "Forschung aktuell" from Deutschlandfunk and team member Paula Mueller. The energetic audience, in which all age groups were represented, soon actively took part in the discussion, asked questions and criticized statements of the discussants on stage.

In his keynote lecture, Prof. Dr. Lars-Oliver Essen tried to convey the scientific context of the evening to the audience. Using various common plants, such as bananas and carrots, he illustrated the products of classical breeding methods through human selection. All the species mentioned above originate from primitive types, which differ from today's food plants, for example by an enormously lower proportion of fruit flesh. He then outlined the differences between more modern breeding methods such as chemical mutagenesis or radiation and genetic tools such as the headline-making CRISPR genetic scissors. At the end of his keynote lecture, Prof. Dr. Essen outlined the ECJ ruling of July 2018 and gave an outlook on topics that influence the genetic engineering discussion, such as the population growth of mankind and the patenting of modern genetic engineering methods as well as the resulting products.

Placeholder image — Fig.1 - Our team member Lars giving the microphone to one of the viewers, that everyone can hear his question.

Most of the panelists agreed that genetic engineering offers opportunities, especially in agriculture, to address challenges such as climate change or the nutrition of the growing world population. According to the introductory statement of Prof. Dr. Andreas Weber from the Cluster of Excellence on Plant Science at Heinrich-Heine University in Düsseldorf, methods of genetic engineering such as CRISPR are already established tools and have no cause for concern, particularly in fundamental research. But subsequently, modified organisms cannot be tested in the field under natural conditions. However, the objective of testing and checking modified plants is a problem, because often independent testing organisations do not have access to certified seeds and the complete data used in the designing process, replied Dr. Christoph Then from testbiotech e.V.

It was also discussed whether the product or the manufacturing process should serve as the basis for evaluation. Since a plant modified by GE methods cannot be distinguished from a plant cultivated by established breeding methods, the product should be the basis of the review, argued Daniel Stukenberg, representative of the party "The Humanists". A point of view with a drastically different background, here represented by ethics professor Dr. Friedemann Voigt, put the consumers in the foreground and demanded their right to know how a food is produced. This led the discussion further in the direction of the effects of genetically modified animal feed on farm animals and the human consumer. At this point, Dr. Christoph Then criticised the sharp decline in risk research, which pursues society's protective goals with regard to health.

The panel also separated into simple gene edited and transgenic organisms. While gene edited plants do not contain genes from foreign organisms, such genes in transgenic plants can, for example, provide certain ingredients that make them resistant to pests or insecticides/herbicides. Transgenic plants must be assessed with special attention and in each unique case, since the individual composition of the ingredients is specific for each modified organism. This point also met with the approval of the audience, which was expressed by strong applause. Transgenic plants could not develop in a natural way and should therefore not be used in agriculture. At the moment, however, the use of genetically modified plants in organic farming is also not in compliance with regulations, according to Freya Schäfer of FiBL (Research Institute of Organic Agriculture).

Prof. Dr. Friedemann Voigt continued to ensure the ethical part of the discussion. He noted that compared to the rest of the world, there is a certain mistrust towards genetic engineering, especially in Europe. One had to estimate the risks against the opportunities, whereby he mainly identified an acceptance problem in the population, which could not be met scientifically. However, there is not only distrust of genetic engineering, but also of the economic motivation to use it. Dr. Claus Kremoser expressed concerns about seed companies with patent monopolies that could promote large monocultures. Nevertheless, the development and application of genetic engineering is strongly dependent on the respective motivation: patents are the driving force in the industry while in the scientific community it is the pressure to publicise. Too strict and thus very costly requirements for the testing of genetically modified organisms make it impossible for small companies to participate in the competition. Consequently, strong regulation would lead to a few large monopolies.

The discussion ended with the consensus that genetic engineering in agriculture should be regulated responsibly and objectively. However, due to ideological blockades and diffuse fears in society and among political influencers, a meaningful willingness to compromise was very low.

Integrated Human Practices

C Y A N O
B I O T E C H

Influencing our project on many levels: growth curves, terminators and well plate cultivation.

Cyano Biotech is one of the leading, top edge companies to work with sustainable and product-oriented phototrophic bacteria in Germany. As a shining figure in cyanobacterial metabolic engineering, the CEO of Cyano Biotech Dr. Dan Enke talked with us about the possibilities of our project. His feedback led us to the design of adjusting specific parameters in our growth experiments and to the integration of our terminator library. Through our talk with a potential end user of our Green Expansion and our engineered strains, we achieved a more “real-world” focus of our project.

As our project was in its beginning, one thing was clear: high throughput methods are essential for any viable chassis in Synthetic Biology. Sadly, we quickly noticed obstacles in our way, such as inhibited growth in our plates compared to flasks. In consequence of that, we looked for help and who would be better, than an expert on that specific field? We talked to Dan Enke and he kindly provided us with data in his own well plate experiments. Soon we noticed, that he inoculated cultures several times in a row out of the exponential phase, leading to a huge boost in doubling times. Through this support we were able to design and conduct a growth curve with as much as five precultures.

Aside from that, we talked about another facet of our project, cyanobacterial terminators. Being overread in cyano´s, both metaphorically and for real, they are one of the most important regulatory genetic elements. Yet another expert in the field of cyanobacteria was pleased of our idea to set on the search for the best terminator and strongly encouraged us to utilize the Green Expansion to its full extent.

Fig.1 - Skype Call with Dr. Dan Enke from CyanoBiotech.

P R O F. D R.
A N N E G R E T W I L D E

Cultivation expertise from leading cyano scientist Prof. Wilde.

While working with the cyanobacterium Synechococcus elongatus UTEX 2973 we noticed very quickly a lack of standardization in the field of Synthetic Biology. To tackle this huge problem, we decided to focus, as one of our main goals, on standardization to make scientific results more comparable. Therefore, we worked on standardizing light measurement, cultivating parameters (temperature, CO2, rpm, …) and the cultivation media for cyanobacteria, especially UTEX 2973.

During the theoretical planning of our project we contacted scientists in research and industry that are specialists for cyanobacteria. As a result, Prof. Dr. Annegret Wilde (Institute of Biology, Albert-Ludwigs-University Freiburg) answered our call and was very interested in advising us in regards to our projects. In order to give us an introduction to the handling of cyanobacteria, she invited us to her institute at the University of Freiburg on 5th and 6th June 2019. In a short and focused internship we were able to quickly gain a set of core competencies regarding sterile inoculation, streaking of cyano cultures and further information regarding the cultivation conditions which we applied to our strain.

In addition, we also learned that the measurement of light intensity is an important topic. There is a variety of measuring instruments and different methods for each, which means that information on light intensities should be viewed with caution. For cyanobacteria such as Synechococcus elongatus UTEX 2973 light intensity plays a decisive role, which is why we analysed differences between a variety of instruments and methods to establish a standard for light measurement based on our results .

In further discussions about our Marburg Collection 2.0 we were recommended to take a very close look at terminators as they oddly enough have an effect on the transcription of upstream genes. As a result, we decided to take a closer look at that and investigated their effects. We thank Professor A. Wilde for her input, her invitation to Freiburg and her recommendations that guided us in our project.

D O U L I X

Confirmation for real case use for our colony picking project.

“My major bottleneck is colony picking.” - Davide De Lucrezia, Managing Director of Doulix

The vision of automating colony picking has existed now for many years. Big companies like Tecan, Singer Instruments or Hudson Robotics invented robots those are able to identify and pick colonies. The problem is that they cost a fortune, starting at 50,000€ up to 100,000€ or even more. Although it is highly desired, a low cost solution is still missing for this tedious task. Start-ups like Doulix are currently automating such workflows in their laboratories, but they do not have the funds to finance a state-of-the-art colony picking robot. By now every single step has to be performed manually, draining resources from other departments, which actually should be paid more attention to. “Colony picking is a bottleneck in every of part our workflows” says Davide De Lucrezia, the founder and managing director of Doulix. Doulix focuses on developing innovative technologies for scientists to simplify their work, especially in the field of Synthetic Biology, and they are currently planning on establishing Opentrons OT-2 in their lab to automate the most part of their workflows.

During an online conference with Davide De Lucrezia, Sota Hirano, and Alessandro Filisetti from Doulix, Davide De Lucrezia suggested that turning the OT-2 into a colony picker as a project would be really interesting. To have a fully trained, ready to use package to turn the OT-2 into a colony picker would enhance the workflow at Doulix tremendously. Nevertheless, to suit the user's needs as well as to get this job done in the spirit of Opentrons, installing the needed add-ons should be as modular and flexible as possible and designed so that “even a biologist” without technical knowledge or programming skills would be able to install and use them.

That is where our team came into play. We decided to take this advice to our heart and started to work out what was needed to turn the OT-2 into a fully automated colony picking robot.

Connections between Opentrons, Promega and QInstruments — Fig.1 - iGEM team Marburg 2019 meeting with Davide De Lucrezia (left), Sota Hirano (middle) and Alessandro Filisetti (right) in a Zoom teleconference to discuss advanced automated workflows in the lab.

One of the first big design questions was whether we wanted to hardcode an image recognition software for the colony detection or if it was a better choice to train a data hungry but - given proper and enough training data - more accurate and scalable artificificial intelligence based colony detection. Kristin Ellis, the director of strategic initiatives at Opentrons referred us to Keoni Gandall from Stanford, a well known tinker of the OT-2 for more unconventional applications. He is building a colony picking system himself, however he chose not to rely on an AI. He recommended us to go with AI as he thinks his approach is very prone to changes in parameters. If many different users want to utilize the same system, a flexible software is required that can take environmental changes into account. We decided to opt for maximum flexibility by working with an AI.

Now that we had an idea of the required software we started to design modular hardware to overcome potential problems in a fully automated workflow in the OT-2. To illuminate the agar plates in the right way without any distortions we engineered a light table that distributes light equally over the plate.

To give an “eyesight” to the OT-2 we mounted a Raspberry Pi 4 and an ArduCAM on the OT-2 arm. For a better accessibility we created our Graphical User Interface for Directed Engineering (GUIDE). We designed our GUI in a way that will enable every user to train their own AI with their own training data set so that the AI can be optimized for each specific situation. Moreover the GUI will also enable access to users that are not trained in computer/ software engineering.

Now that we gave our robot the ability to see, to think and to communicate with us, nothing stood in the way of our own colony picker. We are now able to turn the OT-2 into a colony picker costing below $300. Moreover, for companies, teams or groups who do not own an OT-2 yet, we were able to reduce the costs for purchasing a colony picker by 90-95%, compared to the listed market prices for traditional colony pickers.

Finally we contacted Doulix the second time to discuss in more details about our project. They approved it and gave further suggestions such as “live training” so that the AI will continue to learn as it is being used to pick up colonies. Not only will this improve the AI gradually but it will also adapt to the specific needs of each user. This leaves a lot of room to improve the project in the future.

S T A N D A R D I Z A T I O N

Realizing that we are not the only ones struggeling with no cyano research standarization...

During our visit at the Cyano Conference 2019 in Tübingen we recognized a need for standardization in this community. We asked people to send us their BG-11 recipes and surprisingly received several different versions. The community at the conference was aware of the missing standardization and we received very positive feedback for our efforts. Fixed standards are essential for reproducibility of results, especially the preparation of media and buffers but no one is investing time to set a standard. After the cyano conference we stayed in contact with Nicolas Schmelling, coordinator of the bachelor program at CEPLAS. During his PhD he was working on establishing more standards in the cyano community. He tried to establish protocols and collected different methods and recipes to establish a standard for all.

After he clarified to us the importance of comparable media in context of standards, we started to collect different BG11 recipes and compared them in growing experiments. Figure 1 depicts our results and shows the impact of different recipes for media.
In our experiment we could show that there is a significant difference between the different BG11 recipes despite their relative similarity. During our complete project we were working on the standardization for light intensity, media and different cultivating parameters. We made it our destiny to make the first step into the beginning of standardization in the cyano community by providing Synechococcus elongatus with standardized parameters. We were able to find the optimal growing conditions for UTEX 2973 and could show that creating a standard in measurement and methods is really important to have comparable results. With our project we hope that we could set a first step into standardization, so that the future cyano community will have standardized and comparable results.

C Y A N O
C O N F E R E N C E 2 0 1 9

Knowledge exchange with established cyanobacteria researchers.

E X P E R T O N C Y A N O S :
J A M E S G O L D E N

Defining the absent standardization to discrepancies in light, optical denisty measurement and more.

While diving deeper and deeper into the ocean of possibilities that cyanobacteria have to offer we noticed a few inconsistencies in literature.

BG11 media is commonly used in cyanobacterial research, but the exact composition seemed to be different across every second paper we read. Optical densities are more frequently measured at a wavelength of 730nm, though 750nm seems to be the better choice. For Synechococcus elongatus UTEX 2973 the “optimal growth conditions” according to literature are often quite different; some state 38°C and 500µE at a CO2 level of 3% fits best, others prefer 41°C and 1500µE with 5% CO2 concentration. But how are these light intensities measured? With a planar device or a spherical one? We have not seen this being explained in literature.

As all of these things have an incredibly huge impact on various different experiments we saw the need to find a standardized answer to our questions, reaching out to as many experts in this field as we could reach - whether it was industry or research. One of the leading laboratories working with cyanobacteria is the Golden Lab of the UC San Diego. Susan Golden and her husband James W. Golden have both been working with cyanobacteria for quite some time, now with a stronger focus on their use for industrial purposes. We set up a Skype call with them, but sadly Susan Golden was not able to join us on short notice.

During our talk with James W. Golden we laid open our concerns about the cyanobacterial community and he quickly supported our train of thoughts, as he himself noticed a lack of standardization. He assured us that this is a hot topic in this field of research, as many do not seem to care enough about the reproducibility of their data and encouraged us to continue with our efforts.

More accurately, he talked with us about why he thinks there is still no clear decision on whether to measure optical densities at 730nm or 750nm: It might be a technical problem, as many photometers are simply not able to measure wavelengths of 750nm. In contrast, he mentioned that 750nm would be the more optimal way, as it proves to minimize absorbance from pigments in cyanobacterial cells, presenting more accurate data. This confronted us with a conflicting decision: Would it be better to use the more accurate 750nm or 730nm, as the latter would allow more labs all over the world to measure in the same way.

This was one of the key factors that led us to measure the whole spectrum of our cultures for our growth curves, as this would provide a larger dataset, awarding us not just with 730nm and 750nm data, but also the possibility to check if the spectrum shows normal behavior, from which one could conclude how healthy the cultures are.

JG dif media — Fig.1 - Growth of *S. elongatus* UTEX 2973 at 1500µE measured with different methods.

In the beginning we measured the light intensity of our incubators with a planar measurement device - the only one available for us. Talking to James Golden we realized that we should try to get hold of a spherical measurement device, as he assured us that this is the way to generate more accurate data, leading to a more reproducible setup - exactly what we were aiming for.

After receiving such a device from Biospherical Instruments, we again implemented the feedback we got and measured growth curves. One with cultures at 1500µE measured with a spherical device and one with 1500µE measured with a planar device, where the measured intensities were converted to theoretically spherical values with a conversion chart offered to us by Prof. Dr. Annegret Wilde from the University of Freiburg.

These experiments were a huge step in our project, as they heavily influenced the way we cultured our cyanobacteria, not only drastically improving their growth, but also clearly demonstrating how flawed certain measurements can be. We would never have been able to reach the fast doubling times we achieve now without this crucial input and as this will be the case for others too, we made it our mission to keep on stressing the importance of this way of measurement whenever we reach out to the scientific community. Again, thank you very much Prof. Dr. James W. Golden for your invaluable contribution!

E X P E R T O N C Y A N O S:
N I C O L A S S C H M E L L I N G

And soon, we could add growth media to the growing list of not existing standardization.

O P E N T R O N S
& K E O N I G A N D A L L

Initial inspirations for our colony picking project.

P R O M E G A

Automation of plasmid purification using the OT-2.

When the iGEM year started, we thought about how we could ease the work in the lab using our OT-2. We decided automating the cloning process would be a great idea and soon got into contact with Promega to tell them about our vision. Margarete Schwarz, area manager of southwest germany, and Nans Bodet, a Field Support Scientist (FSS) from the automation department at Promega, were both convinced that the automation of the cloning workflow would be a challenge, but with creativity and some work it would be a major breakthrough and a great tool for everyone with access to an OT-2.

In a Skype call both agreed that they would love to see Promegas Wizard® MagneSil® Plasmid Purification System integrated into the workflow, being Promegas very first automated workflow in Opentrons OT-2 and the first protocol for plasmid purification in a large collection of Opentrons protocols. Promega covered our costs in terms of kits we needed for the protocols so we could focus on optimizing the workflow.

We performed the plasmid purification a few times manually, so we would get familiar with the whole workflow and get a feeling where problems in the automated process could arise. We were in regular contact with Nans Bodet and he gave great advice on how to automate the shaking process in the OT-2 and that we would need the 8-channel pipette to scale up the number of samples that could be handled with our protocol. For the shaker he told us to get in contact with QInstruments, a company which designs and builds small shakers that are simultaneously capable of heating and cooling the samples. Thanks to recommendations from Nans Bodet a member of their support team, Ralf Paetzold, wrote us back and kindly helped us to secure a permanent loan for the BioShake D30-T elm back in June. Through a grant our team won, we were able to purchase the 8-channel pipette arm.

When the shaker arrived, we realized it was a bit bigger than the SPS format for modules in the OT-2 and needed stabilizing support. We designed an adapter for the shaker that is robust enough to withstand the forces that occur during intense shaking.

Furthermore, Opentrons is currently rolling out a major update from their OT-2 3.9 to 4.0 firmware that included a lot of paradigm change. This changed the way we had to define our labware and we ended up defining our shaker module coordinates as a Python dictionary importable via a JSON file. After some calibrations with our OT-2 we were trying to finish the protocol; thankfully Opentrons customer service was patient with us. They told us how to calibrate the OT-2 directly via the terminal because we had some difficulties.

In late august Margarete Schwarz paid us a visit, curious about how the plasmid purification with Promegas Kit would perform and look like in the OT-2. We were also asked to write a blog post about our thoughts and progress on automating plasmid purification for the Promega Connections Blog.

By the end of this iGEM year we were able to develop a working protocol for the single-channel pipette for up to 6 samples, as well as a protocol for the 8-channel pipette for up to 48 samples.

We are very happy about this fruit bearing interaction, we think both sides profited from this cooperation in a big way.

Team:Marburg/Human Practices

H U M A N P R A C T I C E S

Report on Genetic Engineering

G M O R E P O R T

GMO Report

D R . N I N A S C H E E R

Dr. Nina Scheer

P L A N T M A R K E T

Plant Market

P A N E L D I S C U S S I O N

Genetic engineering panel discussion report: Blessing or curse

Integrated Human Practices

C Y A N O B I O T E C H

Cyano Biotech

P R O F. D R. A N N E G R E T W I L D E

Cultivation expertise from leading cyano scientist Prof. Wilde

D O U L I X

Doulix

S T A N D A R D I Z A T I O N

Standardization in Cyanocommunity

C Y A N O C O N F E R E N C E 2 0 1 9

The CYANO Conference 2019 in Tübingen

E X P E R T O N C Y A N O S : J A M E S G O L D E N

Expert on Cyanos - James Golden

E X P E R T O N C Y A N O S: N I C O L A S S C H M E L L I N G

Expert on Cyanos - Nicolas Schmelling

O P E N T R O N S & K E O N I G A N D A L L

Opentrons + Keoni Gandall