Team:Marburg/Human Practices
Human Practices
The most important task of science is to be understood. A finding may be of good quality, but if it cannot persuade society, those qualities will go unnoticed. New technologies cannot exist in our minds alone. They must be accepted.
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Given the numerous opportunities for practical application of cell-free systems in the near future, we have addressed the obstacles faced by the development and application of GMOs. To capture and overcome these obstacles, we have focused our attention on what we consider to be the two most important areas: Science Communication and Education.
We held discussions with two NGOs The Eco-Progressive Network (PAW) and the German Farmers' Association (DBV) to better understand the farmers' views on GMO use. During the discussions, it became clear that one of the main reasons for the widespread resentment within the public is linked to a lack of knowledge. Considering the statements of the representatives of the farmers' associations, we put special emphasis on education. That is why we have produced an Infolet with information on green genetic engineering, aimed specifically at farmers.
To improve the education of highschool students, we have collaborated with the company miniPCR to introduce cell-free systems as part of the so-called BioBits kits in biology courses. We have also set the course for a long-term cooperation between GASB and schools, in which the schools can present cell-free systems in class every year.
Biocontainment is another aspect that we have given special attention to because of the explosive nature and relevance of the topic.
We evaluated the statements of the Central Commission for Biological Safety,the European Food and Safety Authority and the responsible Hessian Ministry and came to the conclusion that many guidelines and laws are outdated or contradictory, which can be seen as yet another reason for the lack of science communication.
A special part of our science communication project was the discussion panel with the Children's and Youth Parliament, which represents all of Marburg's students. In the follow-up discussion, we came to the conclusion that young people are basically very open and positive about genetic engineering, as long as the scientific background is explained in an appropriate way for the target group.
Since we are confident that OpenPlast will be a major advancement for synthetic biology, we sought out contact with users and researchers in the field of green engineering and integrated the feedback we received back into our project.
To incorporate their opinions and priorities into our laboratory research, we held discussions with representatives of leading seed companies from Germany (KWS) and the U.S. (Corteva). They stressed the need for our team to show a direct comparability between our prototyping platform and in vivo results. This led the team to focus on the task of chloroplast transformation to compare with in vitro data.
Our project is designed as a rapid prototyping platform, therefore we got into contact with researchers in academia. A key finding from discussions with Lauren Clark and Michael Jewett at Northwestern University is to identify endogenous transcription as one of the limiting factors for the application of cell-free systems. To further improve our project and make it useful for real-world applications in labs around the world, we set out to make endogenous transcription work in our extracts.
NGOs
NGOs offer an interesting perspective on our project, not only regarding the future use of GMOs, but also in terms of public perception and the lack of knowledge about synthetic biology in this context. For this, we met with the Eco-Progressive Network (PAW) and the The German Farmers’ Association (DBV). In the meetings, we realized the need for the general society and especially farmers to require a more profound knowledge on the topic of GMO's to overcome prejudices and be able to engage a productive discourse.
Education
To establish content that
could be distributed and reproduced, even without our iGEM team’s involvement, we decided to create a
comprehensive booklet. This “Infolet” was written based on the feedback we got during the interview with the
farmers. This Infolet features articles about the history, biocontainment and legal as well as
scientific questions regarding green genetic engineering in Germany.
Students form the very foundation of any future development in our society. Thus, in the second part of our
educational project we built up a long-term improvement of the German curriculum for high schools. In cooperation with the German Association for Synthetic Biology (GASB) we first tested and will now
provide BioBits kits by miniPCR for high schools to be able to teach molecular and synthetic biology already at an early level of education.
ZKBS
Building up trust and acceptance is especially for green genetic technology an ongoing challenge to which we want to make an impactful contribution with our project. To tackle the existing problems, we sought the advice of Dr. Jan-Wolfhard Kellmann, deputy member of the ZKBS (Central Commission for Biological Safety), at the beginning of our work. In a personal interview he gave us the insight that not only scientific gaps have to be filled, but that our project will also face major political barriers.
Our work in Human Practices is primarily guided by this and extends to our progress in Education/Communication. In order to meet our aspirations, we wanted to interact with a range of policy levels, from international to local, to identify how OpenPlast will be able to contribute to a better future.
EFSA
The European Union is the foundation of the international legal situation in Europe. We had the opportunity to contact the European Food and Safety Authority (EFSA) and ask them about the impact of our technology in the approval process of new GMOs in the EU. By using chloroplasts we see a realistic simplification of the approval hurdles confirmed with the criteria mentioned by EFSA.
Hessian Ministry for the Environment, Climate Protection, Agriculture and Consumer Protection
We found the greatest challenges at the national and regional levels of politics. Priska Hinz has been leading the Hessian Ministry for the Environment, Climate Protection, Agriculture and Consumer Protection for over seven years. With her help, we were able to understand the political decisions against green genetic engineering in Germany. We found that existing policies are directly linked to an impression of aversion by the general public and stakeholders from agriculture. We can change this! Therefore, we talked directly with different farmers in the context of Education/Communication and wrote in joint efforts a comprehensive booklet about genetic engineering.
Lord Mayor
The city of Marburg is characterized by its deep interweaving of university and social life. Our team sees the management by local politics of the interaction between the scientific campus and the population as a prime example of which interfaces promote acceptance among those two groups. Therefore, we conducted an interview with Thomas Spies, the mayor of Marburg. Here we focused on better education as well as building a bridge between academical research and economy, which we want to encourage through liberalization in the development of green genetic engineering with the help of our Cell-Free Systems.
Children and youth parliament (KiJuPa)
In times of upheaval, when the future is in question due to climate change and globalization, children are the best judges about decisions for tomorrow. With this philosophy, we sought a dialogue with the Children's and Youth Parliament (KiJuPa). Based on our experiences at the Dies Academicus information event, we designed a presentation aimed at children and young people to provide an overview of green genetic engineering. Afterwards we opened an open discussion on this topic with the members of the KiJuPa. As a conclusion we saw our view strengthened that the youth of Germany has an open ear for realizations of biology and genetic engineering. Thus, with sustainable improvement of education through our cooperation with GASB and MiniPCR, we aim to overcome some of the general aversion against GMOs.
Integrated Human Practices
We are confident that OpenPlast will provide a major advancement for synthetic biology and accelerate further research. We dedicated our time to gather feedback on which criteria our laboratory research has to excel, to tap the full potential of the project.
Industry
From the very beginning we understood the value of having input from researchers who actually work on a day-to-day basis in plant biotechnology. If OpenPlast was to ever become more than just an iGEM project, we would have to create something that addresses the true needs of agricultural industry sector.
To implement their opinions and priorities into our laboratory research, we aimed to interview leaders in the area, namely seed companies from Germany (KWS) and the US (Corteva) and gain perspective in their current workflow, their viewpoint on transplastomic plants and to ask them a simple but crucial question: "What experiments would you want to use our cell-free system for?"
Both Dr. William Gordon-Kamm from Corteva Agriscience and Dr. Klaus Schmidt from KWS SAAT emphasized that our team needs to show a direct comparability between our prototyping platform and in vivo results. This prompted the team to focus on the laborious task of chloroplast transformation with the intent of comparing it to the in vitro data
Academia
Northwestern
University:
Our project is designed to be used as a rapid prototyping platform, with academic
researchers as our primary target group. Thus, we wanted to reach out not only to researchers in the
industry, but also in academia. One specific question we wanted to explore is why chloroplast in vitro
systems are not used more commonly nowadays, since first advances to such systems have already been made
decades ago.
Talking to Lauren Clark and Michael Jewett from Northwestern University, we learned that
endogenous transcription is one of the main limiting
factors. To further improve our project and make it more useful for real-world applications in labs around the
globe, we therefore set out to robustly establish endogenous transcription in our extracts.
Apart from this
crucial information that we gained, this interview was incredibly helpful for us, as we were able to learn a lot
about cell-free systems and what to keep in mind when working on them. We are very grateful for these inspiring
conversations and are happy that we were able to gain so much from it for our project!
Interview with Northwestern University
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Implementation
Selection marker Screening
Some plants are not used in academic research and industry because it of the difficulty to transform and genetically modify them. This puts a huge obstacle in the way of our vision of a transplastomic approach to transgenic plants. After our exchange with KWS, we had the necessary feedback to tackle this issue.
One of the challenges of transforming the chloroplast lies in the copy number of the chloroplast genome. During the primary transformation process only few copies are transformed, meaning that the transformed cells contain wild-type and transformed versions of the genome, which is called heteroplasmy. In order to propagate the transgenic plastid genome, the cells have to be kept under continuous selection pressure, until the homoplasmic state is achieved - the point when all genome copies are transformed. This task seems even much more challenging for seed plants, because a typical leaf cell contains 1000-2000 copies of the plastome. If the chloroplast genomes are still heteroplastic and the selection pressure is removed, there is even the possibility of reverting back to the wild type chloroplast genome without the transformation cassette.
In order to test different antibiotics as selection markers in vivo, which would cause enough selection pressure, genetic parts need to be characterized to express the desired selection marker , but the genetic parts cannot be characterized without obtaining successful transformants. In order to solve this chicken or egg problem, our cell-free systems come in.
With OpenPlast, parts can be directly added and expressed by the native transcription/translation machinery, without the need of transforming a living cell. Therefore, expression level of parts can be evaluated with high throughput. In addition to that, it allows for screening of new selection markers. Based on the feedback from the interviews, we performed an experiment in which different antibiotics were used to inhibit the protein biosynthesis in the cell-free systems, whereby strong inhibition of protein biosynthesis would point to a suitable selection marker.
As displayed in the graph, we screened for different selection markers in N.tabacum and T.aestivum. Most antibiotics inhibited protein biosynthesis in both tobacco and wheat. In this respect, these antibiotics could be tested with a corresponding resistance in the respective plants.
Ampicillin has an inhibitory effect on cell wall synthesis in bacteria, but since chloroplasts do not have a cell wall, the extracts should still be able to carry out protein biosynthesis and luminescence should be seen. This was exactly what was observed in the experiment. Furthermore, high expression can also be observed in wheat when spectinomycin is added. T.aestivum has a resistance to spectinomycin, contrary to tobacco. These results demonstrate the possibility of inhibiting protein biosynthesis in the cell-free extracts using antibiotics and, together with resistance marker genes, can be used for targeted selection.
If successfully established, novel selection markers could therefore enable the selection of homoplasmic tissue cultures in a wider variety of plant species, making the process of engineering them much easier to achieve.
Endogenous Transcription
In our early measurement constructs we made use of a promoter from a T7 DNA polymerase and a corresponding T7
promoter from the T7 bacteriophage, as they provided high expression levels for our luminescent proteins and they
are commonly used in cell-free systems. Using the T7 polymerase in transplastomic plants has serious downsides,
though.
It causes strong phenotypes and growth defects, limiting the choice of promoters to endogenous ones.
It is, however, extremely difficult to characterize them in cell-free systems: their expression values are so low
that it’s not detectable by most assays. In spite of these hurdles, our conversation with Lauren Clark evidenced
the importance of having them in our toolbox, as they would allow us to create larger metabolic pathways, bringing
chloroplasts synthetic biology to a whole new level.
This motivated us to focus our efforts in optimising our CFS for endogenous transcription. In bacteria is a dialysis necessary for native promoters to function, but this isn’t necessarily the case in chloroplasts, we often just need to adapt the Magnesium concentration, according to Lauren Clark.
Finally, gene expression was significantly increased by optimizing the methods used to lyse the chloroplasts. In
the end, OpenPlast was fully capable of performing protein biosynthesis using endogenous transcription and
translation!
After the optimization of the endogenous expression, several promoters were characterized in the
CFS.
The graph below shows that tested promoters do not only result in high expression, they even show different expression levels and can be used for precise engineering in genetic circuits. The PpsbA Promoter performed the worst and Prrn 16 Wheat Promoter performed the best.
in vivo Experiment
Another valuable input came from Dr. Gordon-Kamm, from Corteva, laid out the importance of presenting in vivo experiments as well. Despite the limited time we had to perform the transformation, the team agreed with the advice of Dr. Gordon-Kamm and worked tirelessly to finish the experiment in time. Tobacco plants were transformed with four different constructs for the characterization of 5’ UTRs (psbB, pabC, rps2, clpP mAAGA (-20 to -10) , which could then be compared with in vitro data.
Further information on the implementation of feedback can be found in the sub-pages of the individual interviews.
Conclusion
In conclusion, we reached out to the end users of OpenPlast with the intent of gathering a variety of opinions and viewpoints on what would be the main requirements for the implementation of cell-free chloroplasts as a prototyping platform on a wider scale. They not only contributed with their perspectives on the current state of the plant biotechnology industry and where it is going, but also provided valuable insight on where our efforts could be focused to achieve our goal. We successfully implemented these ideas during the course of the project, showcasing OpenPlast as an attractive alternative to the traditional method of chloroplast transformation.