Team:TU Darmstadt/human practices

Integrated – TUDA iGEM 2021

Integrated

Science is and has always been of social importance and consequently has to be handled as a social matter. For this reason, Human Practice and its important component Integrated Human Practice (IHP), described by iGEM’s director of judging as “The study of how your work affects the world, and how the world affects your work.”,​1​ is an essential part of iGEM. Its mission is to identify the impact of a scientific project on the society and our world.

In order to do so, it is of utter importance to consistently assess how your project is received by the general public and by relevant stakeholders. Their input, whether it is fear, advice or approval, needs to be included into the evaluation and the execution of our project.

Our team made an extensive effort to evaluate the impact of our project PHIRE BYRD on the society and the global community. For the evaluation we relied on the input from the general public, scientists, ethicists and government officials.

Our Approach

The CEA Framework:

To ensure that our work and its presentation are structured, we looked at different frameworks which we could use as an exemplary model for developing our own strategy.

We found the framework created by Professor Richard Owen​2​ for responsible research and innovation, that has already been used by the iGEM Team Exeter in 2017,​3​ called AREA​4​ most suited as an inspiration for the creation of own simplified framework.

The AREA framework continuously seeks to:

Anticipate the intended or unintended consequences that might arise;

Reflect the purpose, motivations and potential impacts as well as the possible risks;

Engage in a dialogue or debate about the project in an inclusive way and;

Act according to the input on the project.​4​

For our own framework we adjusted the AREA framework and the namesake CEA framework to fit our requirements. The is implemented in many management methods, most prominently in the agile project management​5​ stands for City Energy Analyst and is a computational structure for the examination and enhancement of energy frameworks in areas and city regions.​6​ We call our framework CEA as well but in our case it stands for Consider, Evaluate and Adapt (Figure 1).

Figure 1. Schematic overview of the CEA framework. CEA stands for Consider, Evaluate and Adapt and it was used to structure and present our work in Integrated Human Practices. This framework describes the system behind the assessment process of the impact on the society and the world of our project PHIRE BYRD.

With Consider the steps “Anticipate” and “Reflect” of the AREA framework were combined. In it the potential areas of impact, positive and negative, of our project PHIRE BYRD as well as potentially relevant stakeholders were mapped out. During the Evaluate part the main goal was to engage with the relevant stakeholders by scheduling and conducting interviews with scientists, ethicists and government officials and by performing a survey for the general public. All this in order to gather outside input (feedback, advice, etc.) on our project and to evaluate how to further develop it. The final step Adapt considered what had been gathered from the first two steps of the framework. Here it was necessary to decide how we wanted to pursue our project, i.e., determine based on the given input which detail shall be changed and which shall not. During this phase of the cycle, we might also have encountered new areas and aspects of our project we hadn’t considered before. Due to this a new cycle started.

Main Results of Our Work

  • We learned that we do not need to worry about lactonases degrading our AHL but should adapt our wet lab design to test for cytotoxicity with a wider range of AHL concentrations.
    • Based on the interview with Prof. Dr. Fabian Commichau
  • We found out that it is highly likely for our AHL to diffuse through the Gram-positive cell wall of Bacillus subtilis and how to design an experiment to confirm it.
    • Based on an e-mail from Prof. Dr. Anke Becker
  • We embraced the social responsibility scientific work carries and aligned our Human Practices with the Responsible Research and Innovation (RRI) framework of the European Union.
    • Based on the interviews with Prof. Dr. Alfred Nordmann
  • We learned how to orientate both mutant lox sites to achieve an inversion of our promoter cassette.
    • Based on the interview with Prof. Dr. Beat Lutz
  • We found out that the rrnB terminator is a strong and universal terminator for the termination of the transcription of any Bacillus gene, which is why we used rrnB terminators within our cassette design for working in Bacillus subtilis.
    • Based on an e-mail from Prof. Dr. Jan-Willem Veening
  • We redesigned our genetic circuit for the phage release using RecA730.
    • Based on interviews with Prof. Dr. Grzegorz Węgrzyn

In this part of our page, we will guide you through our conducted interviews.

Being Introduced to IHP

Meeting with Dr. Sascha Vogel – Science Birds GmbH

Dr. Sascha Vogel is the founder of the Science Birds GmbH.

The Science Bird GmbH is company full of young and motivated scientists who enjoy bringing their enthusiasm for the wonders of science to the world by holding workshops on concepts of scince, soft-skills you need in the industry, etc. .​7​

His career in science communication is built up on his academical experience in physics.

Dr. Sascha Vogel helped us kickstart our IHP work with a workshop about Science Communication. The main learnings we took with us for our work in IHP were:

  • Being open for different experiences and willing to adapt
  • Gathering input from as many stakeholders as possible using different methods as you cannot talk to an ethicist and a scientist in the same way
  • Making science accessible and understandable for everybody and not only a limited amount of people

After this we embarked on our journey of integrating opinions and advice of different relevant stakeholders.

Expert Talks

How Integrated Human Practice affected…

the scientific approach of the project:

Prof. Dr. Fabian M. Commichau

Prof. Dr. Fabian M. Commichau is a professor at Brandenburg University of Technology Cottbus-Senftenberg.

His special field is synthetic microbiology and his research focuses on second messenger-mediated signal transduction and mechanisms of gene regulation in Bacillus subtilis.

Why did we talk to him?

Initially we wanted to talk to him about the possibility of AHLs to enter Gram-positive cells, but in the end we were able to draw further conclusions from our talk with Prof. Commichau. 

What information did we gather and how did we adjust our project?

We were wondering if AHLs, which are typical quorum sensing molecules for Gram-negative bacteria like Pseudomonas aeruginosa, could get into our Gram-positive Bacillus subtilis sleeper cell. AHLs can have a cytotoxic effect on Bacillus subtilis (B. subtilis) in certain concentrations, therefore lactonases are expressed in some strains to degrade AHLs. Because our system depends on the presence of AHLs inside B. subtilis, it was important for us to get more information about these aspects.

Prof. Commichau told us that AHLs could enter B. subtilis through unspecific lipid transporters besides diffusion. He also referred us to Prof. Dr. Anke Becker as an expert for AHLs, who we also contacted regarding this. Concerning the degradation of AHLs through lactonases, Prof. Commichau explained that these enzymes are mostly only active under certain conditions. Therefore, lactonases should not have a great impact on our system. To test the cytotoxic effect of AHLs, Prof. Commichau recommended us to test higher concentrations of AHLs than we had initially planned. In this way we could see whether there is a formation of mutants.

In the lab we only tested our sensing circuit in Escherichia coli and not in B. subtilis. Because of that, during our talk we also discussed the transferability of our genetic constructs into B. subtilis. Prof. Commichau told us that in principle our circuit tested in E. coli should also work in B. subtilis. But we must consider adapted promoters for B. subtilis, ;and we should also optimize the codon usage of our constructs for B. subtilis. We implemented his advice in our theoretical lab plan.

Prof. Commichau also made some interesting suggestions concerning potential applications of B. subtilis biofilms and therefore our sleeper cells. He told us that B. subtilis is often used in sanitary facilities, where it could help to kill and suppress human pathogens.

Moreover, our sleeper cells could be useful at the root area of plants. Here, our sleeper cells could be engineered to target different plant pathogens, like Xanthomonas species.  

All in all, Prof. Commichau helped us a lot to realize and adapt our genetic circuit in B. subtilis and proposed many interesting areas in which our sleeper cell could find application.

Prof. Dr. Anke Becker

In the course of our research our attention was brought to Anke Becker who is a PhD of microbiology and PI as well as the director at the Centre for Synthetic Microbiology at the Philipps-Universität Marburg in Germany. Additionally, Prof. Becker is an editor or associate editor of Journal of Bacteriology, Journal of Biotechnology and BioDesign Research.

Why did we talk to her?

A crucial part of our project is being able to sense pathogens intruding the biofilm of interest. Since in our example the pathogen is Pseudomonas aeruginosa (P. aeruginosa) we decided to modify the genetic circuits published by Ying Wu (2021) and Nazanin Saeidi (2011) for Bacillus subtilis (B. subtilis) instead of Escherichia coli (E. coli).

We had to consider that the cell walls of Gram-positive bacteria (B. subtilis) are thicker than the walls of Gram-negative bacteria (E. coli). This might be problematic for the quorum sensing molecule of P. aeruginosa (AHL) to enter the cell and therefore to trigger the genetic circuit. Another expert we talked to, Anna Dragos, made us aware of this problem.

As a consequence, we wrote to Anke Becker for her expertise concerning quorum sensing in B. subtilis.

What information did we gather and how did we adjust our project?

She told us, that she does not know of a case where it was tested if AHL molecules could diffuse through the B. subtilis cell wall. Nonetheless, in her expertise, she thinks it is highly likely that short-chained AHLs (C4 – C8) as well as modified AHLs which are more hydrophilic can pass through the cell wall. Ms Becker based this assumption on a marine Exiguobacterium (Gram-positive) producing and sensing AHLs. Since the AHL used in our project is modified it is more hydrophilic.

One possibility to make sure that Ms Becker´s assumption is correct is to put a reporter gene under the control of a LuxR dependent promoter and add the compliant luxR gene under the control of a constitutive promoter. By adding AHL into the medium it can be tested if a diffusion through the cell wall would take place. Also different LuxR/pLux pairs can be used to test various AHLs.

Additionally, Anke Becker brought our attention to the question, if our genetic circuit would be sensitive enough to measure the concentration of AHL produced by P. aeruginosa. This could especially be problematic, because most microbial communities produce AHL degrading enzymes.

Dr. Anna Dragoš

Dr. Anna Dragoš is an Assistant professor at the University of Ljubljana in Slovenia.

Her research is based on bacteriophages, their interaction and influence on bacterial colonies. Her work specifically focuses on Bacillus subtilis.

Why did we talk to her?

We talked to her becuase being an expert on both phages as well as B. subtilis, more than qualified her to help us to establish our self-developed project concept. Dr. Dragoš provided a lot of input about the integration sites in Bacillus cells and the transformation process.

Which information did we gather and how did we adjust our project?

Firstly, Dr. Dragoš recommended using a natural competent B. subtilis strain like 168, so that we can efficiently transform our B. subtilis cells with the DNA of the lambda phage. Secondly, we went over the topic of ensuring a stable integration of the phage´s genome in the cell. Therefore, she proposed to consider the amyE locus, which is a locus widely used to overexpress different genes. Also, she mentioned that using a region near the replication termini, where B. subtilis prophages, e.g. spß prophages, are integrated, would be a promising possibility. Thirdly, we further talked about the phage release. Dr. Dragoš made us aware of the fact that holins are highly specific, which means that our phage release through B. subtilis holins should be successful.

Prof. Dr. Grzegorz Węgrzyn

Prof. Dr. Grzegorz Węgrzyn is a professor at the Department of Molecular Biology at the University of Gdansk in Poland.

His research on phages is focused on the lambda phage and its genetic switch.

Why did we talk to him?

Being author of a book related fully to the lambda phage, made it really beneficial for us to talk to him in order to get answers on very specific questions. He helped us with our concept for the phage induction and he also contributed on the discussion about possible challenges that our project might have to face. Furthermore, Prof. Dr. Węgrzyn provided us with the needed Escherichia coli lysogen strain and protocols for our experiments.

Which information did we gather and how did we adjust our project?

Prof. Dr. Węgrzyn described our project concept as “challenging but possible” as we aim the expression of phages by a self-developed genetic switch, which should be inserted in bacterial cells that are not the natural host organisms. Firstly, he presented us possible ways on natural induction of the lytic cycle e. g. by UV-light. Secondly, we discussed about possible ways to design our controlled genetic switch. We wanted to stop the expression of cI in the presence of pathogens and this way activate the production of new phages, as the concentration of the repressor would reach minimal values. Prof. Dr. Węgrzyn explained that this concept would most possibly be very slow, as cI is a very stable protein. Therefore, he mentioned another possibility, using the SOS response that naturally causes the cleavage of the cI and thereby induces the lytic cycle. He proposed the RecA mutant (RecA730), as it does not require to be activated via DNA damage. The knowledge on this mutant, allowed us to design a faster and more promising switch to induce the phage expression. Moreover, Prof. Dr. Węgrzyn noticed that the cI cannot be cleaved by RecA in the thermosensitive mutant. For this reason we decided to use the thermosensitive mutant only as a positive control for the phage induction. Lastly, he made us aware of the fact that having an E. coli optimized phage in B. subtilis could lead to a weak phage expression. Even though our experiments are based only in E. coli, we took his feedback under consideration as shown in the Outlook & Challenges.

Zihao Yu

Zihao Yu is a graduate student and researcher in the laboratory of Dr. Lanying Zeng at the Texas A&M University.

He focuses on the lambda phage and its switching mechanisms between the lytic and the lysogenic state.

Why did we talk to him?

We talked to him in order to gather input to help us design our genetic circuit for the phage production upon induction.

Which information did we gather and how did we adjust our project?

Our proof of concept is based on the lambda phage. Talking to Mr. Yu was a great opportunity for us to gain a deeper understanding of the lysis-lysogeny switch in enterobacteria phage. Also, discussing which factors we could use to integrate the switch in our own concept was an important step for our project development.

Methods for modification of the lambda phage genome were also part of the meeting. This has helped us evaluating possible difficulties when engineering the genetic circuit. Mr. Yu agreed with our idea to use a direct output, e.g., a single gene controlled by our switch. Thus, we developed our proof of concept using the reporter gene eGFP to test the functionality of our switch. All in all, the meeting gave us a new perspective regarding the work on the lambda phage and its induction.

Dr. Jason Gill

Dr. Jason Gill is an associate professor at the Texas A&M University.

His research is focused on bacteriophages, especially their genomics as well as possible applications.

Why did we talk to him?

We talked to Dr. Gill to critically reflect our project. Especially by discussing bottlenecks and possible solutions.

Which information did we gather and how did we adjust our project?

We discussed the choice of the lambda phage as our proof of concept and our RecA mediated switch to control phage production. Thus, we make usage of RecA as a coprotease that is produced during the SOS response in E. coli. Dr. Gill explained that LexA is also a well-known protein of the global stress response of E. coli. Naturally, RecA causes the cleavage of LexA. Since the repressor cI and LexA are similarly structured, RecA also induces the cleavage of cI. This means that by cleaving cI, which is crucial for the lysogenic cycle, we could induce the production of lytic phages. Dr. Gill also told us that even B. subtilis has a RecA homologous, which would be a great linkage between our E. coli based proof of concept and our bacterial chassis B. subtilis. Moreover, we discussed challenges we are facing, e.g., the integration of the phage in our bacterial chassis. Dr. Gill encouraged us in our approach of using a quorum sensing induced phage production. This could help improving the modularity of our system, allowing a protection of biofilms against various pathogens.

Dr. Wadim Weber

Dr. Wadim Weber is a postdoc at the Technical University of Darmstadt.

He has focused his research on nanopores and their particular applications. Thus, he has been working on holins, which attack the inner cell membrane.

Why did we talk to him?

We talked to him in order to find out more about holins, their working mechanism and their importance for the phage release that is a key aspect of our phage-defense system.

Which information did we gather and how did we adjust our project?

Dr. Wadim Weber helped us with our concern regarding an unexpected destruction of holins. He told us that there are probably no cases where bacteria could develop a defense mechanism against holins. However, he mentioned that a rarely occurring phenomenon could be the destruction of the proteins before they can complete their function inside the cell.

Moreover, we discussed about the antiholin-holin system, the “inner hour” of the phages, which works by controlling the time of infection. This system is based on the action of the antiholin, which “blocks” the further formation of the holins. Hereby we could modify the holins, and achieve a controlled release of a precise amount of phages through the pores with an adjusted size. This is beneficial in order to avoid a very early prophage release that may cause a weak infection of the targeted bacteria. In our case this would result in a weak defense of the biofilm.

Furthermore, Dr. Wadim Weber told us about three types of holins the S21-68, S105 and the T4:

  • The S21-68, known as the pinholin, creates very small pores, which is not suitable for our application since we want to release the whole phage.
  • The S105 creates pores up to 1 μm and allows the endolysins to go from the cytoplasm to the periplasm and further disrupt the cell.
  • The specificity of the T4 holin is its ability to fully lyse a cell without the necessity of lysins. For this reason, T4 causes very high lysis rates.

All in all, he suggested us not to focus on the antiholin system because simpler options are possible. Thus, he proposed the modification of the promoters responsible for the holin expression as another way, to modify the cell lysis timing. Based on our aim of a successful phage release, Dr. Wadim Weber recommended to work either on S105 or T4.

Prof. Dr. Beat Lutz

Prof. Dr. Beat Lutz is a faculty member at the University Medical Center of the Johannes Gutenberg University Mainz in Germany.

His research focuses on lipid signaling systems in the brain and peripheral organs.

Why did we talk to him?

We talked to him because even though his current research focuses mainly on lipid signaling systems in the brain and peripheral organs, he has employed the Cre-Lox system on previous investigations. He elucidates about this system in his research article “Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein“.

What information did we gather and how did we adjust our project?

Prof. Dr. Lutz helped us correct the orientation of the lox sites in our kill-switch proof of concept construct and confirmed that we were employing the correct mutants (lox66 and lox71). He also went through our construct and corrected the orientation of the promoters so that they would be inverted in the right way. Additionally, he recommended us to use qPCR to quantify the inversion.

Prof. Dr. Jan-Willem Veening

Prof. Dr. Jan-Willem Veening is a professor at the Department of Fundamental Microbiology at the University of Lausanne in Switzerland.

His specialties are systems and synthetic biology approaches to study Pneumococcus cell biology, the development of antibiotic resistance and phenotypic variation and its importance for virulence.

Why did we talk to him?

We talked to him because while his current focus is Pneumococcus, he utilized B. subtilis as a model organism for his PhD dissertation research on phenotypic bistability.

What information did we gather and how did we adjust our project?

Prof. Dr. Veening helped us establish that rrnB terminators could be used for termination of all B. subtilis genes within our theoretical constructs and answered our doubts regarding codon optimization in B. subtilis, which was of vital importance for the design

Dr. Thomas Gorochowski

Dr. Thomas Gorochowski is a Royal Society University Research Fellow at the University of Bristol and an expert on the field of synthetic genetic circuits.

Why did we talk to him?

We talked to him because we saw similarities in the way our kill-switch construct works and a computational AND-Gate, which he is an expert for.

What information did we gather and how did we adjust our project?

Thanks to his valuable help, we were able to correct our initial assumption and develop a circuit model that reflects the way our kill-switch works. Moreover, Dr. Gorochowski also helped us understand the limitations of our model and to develop an assay design to test whether our assumptions about the Boolean character of our construct are true.

Dr. Ilka Bischofs

Dr. Ilka Bischofs studied physics at Würzburg, Stony Brook and the Max Planck Institute of Colloids and Interfaces.

She got interested in bacterial stress responses and signalling systems and gained postdoctoral experience in the Arkin lab at UC Berkeley and the Sourjik lab at the ZMBH.

Since 2016 she is affiliated with the Max Planck Institute for Terrestrial Microbiology and heads the department-independent research group Complex Adaptive Traits (µCATs) at the BioQuant. Her lab uses tools from systems- and synthetic biology to understand, control and engineer the adaptive behaviors of beneficial Bacilli.

Why did we talk to her?

We talked to her to get information about the following topics: the cultivation of a biofilm and what strains she could recommend for our project, the cultivation of a co-culture and the homogeneity of the biofilm.

What information did we gather and how did we adjust our project?

Based on her recommendations and her advice, we decided to switch from our initial strain of choice – 168 – to only using the biofilm forming strain NCIB3610. Due to her advice concerning the cultivation of a co-culture we decided to suspend further research into the creation of synthetic dependencies, at least until we had more data concerning the homogeneity of our co-culture.

Prof. Dr. Ákos T. Kovács

Prof. Dr. Ákos T. Kovács studies bacterial interaction and evolution, and the underlying phenotypic and genetic changes with the aim to use this knowledge towards green and white biotechnology.

His group aims to reveal the ecology of Bacilli, understand their social interactions during biofilm development and adaptation in the presence of other microorganisms, including soil derived bacteria and fungi, and eventually to their natural niche, the rhizosphere of plants.

Why did we talk to him?

We spoke to him to get input, feedback and recommendations about the following topics: the cultivation of a biofilm and what strains he could recommend for our project, the cultivation of a co-culture and the homogeneity of the biofilm and our lab plan including methods and assays to identify potential sources of errors.

What information did we gather and how did we adjust our project?

Based on his recommendations and advice we planned to cultivate both a co-culture of two differently genetically modified B. subtilis cells from the same strain NCIB3610/DK1042 as well as a co-culture of B. subtilis cells from two closely related strains like NCIB3610/DK1042 and 168 a multi-strain consortium. Further we extended our plan for the lab, with the confirmed co-culture cultivation procedure, the transformation protocol and the advice on the right moment (after 2-3 days on Msgg at 30°C) for the implementation and the method for the evaluation of our assays. We were also able to reproduce the assembly of the two plasmids phyGFP and phymKate2 containing the fluorescence markers thanks to Dr. Kovács’s plasmid maps and his recommended guiding papers: Gestel et al. (2014) and Veening et al. 2009. Lastly, only thanks to him providing us with the B. subtilis strains (B. subtilis DK1042, B. subtilis TB34, B. subtilis TB35) as well as the transformed E. coli strains (E. coli MC1031 containing plasmid phyGFP and E. coli MC1031 containing plasmid phymKate2), we were able to start our proof of concept in the lab.

Prof. Daniel Kearns

Professor Daniel B. Kearns from the Indiana University in Bloomington, USA is an expert for the domestication and biofilm formation of Bacillus subtilis.

Why did we talk to him?

We talked to him to discuss our project, especially our lab plan with Prof. Kearns to receive expertise on the work with B. subtilis as well as the formation of biofilms.

What information did we gather and how did we adjust our project?

The input with Prof. Kearns helped us to finalize our lab plan and highlighted potential mistakes we could have missed otherwise.
Based on his expertise we decided to switch from our dual strain approach to using only a single strain, to be exact the biofilm forming strain DK1042. The usually used 168 strain of B. subtilis is a descendent of the DK1042 strain, which should ensure that all modifications made to the DK1042 strains should also be feasible in the normally used 168 strain, enabling a high compatibility and transferability of our results.

Prof. Dr. Alfred Nordmann

Prof. Dr. Alfred Nordmann works on philosophy and history of science at the Technical University of Darmstadt.

He is concerned with the interface between philosophy of science and philosophy of technology. On the one hand, he investigates the varieties of objective cognition, on the other hand epistemological, metaphysical, aesthetic aspects of technoscientific research.

Why did we talk to him?

As a professor of philosophy and philosophy of science, Alfred Nordmann has often helped in the past iGEM years. Therefore, we decided to talk to him to get a general overview of the ethical aspects of synthetic biology. In doing so, we wanted to focus on our responsibility to society in order to improve our Human Practices.

What information did we gather and how did we adjust our project?

Technosciences such as synthetic biology are used to solve social problems. As a result, this research is more in the focus of society and its judgement. In contrast to other research areas, the pressure to provide evidence increases in the case of synthetic biology. This is because any use of GMOs can lead to unforeseen consequences. These possible changes to nature lead to reflexive rejection of the society, as they make the world less controllable. Possible damage can only be detected by long-term observation.

For this reason, it is important to provide attractive sources of information for them to get people interested. Fears about GMOs and synthetic biology should be taken seriously in this context.

That is why Afred Nordmann inspired us to align our Human Practices with the RRI concept. The concept of Responsibility, Research and Innovation (RRI) focuses on the integration of scientific and technological progress into society.

Prof. Dr. Kathryn Nixdorff

Prof. Dr. Kathryn Nixdorff conducted research at the Institute of Microbiology and Genetics, at the Technical University of Darmstadt.

During this time, she also addressed questions of responsible research, in particular concerning biosecurity and dual-use problematics.

In 1987 she was a founding member of IANUS, an organization dealing with interdisciplinary peace and conflict research at the Technical University of Darmstadt.

Why did we talk to her?

Our project was to design a modular system that is not only suitable for release, but also for the use in a wide variety of applications. Therefore, it was important for us to learn about crucial points in the development of a generally safe system. But we were not only interested in questions of biosafety, but also of biosecurity. In particular, whether our system allows forms of abuse – e.g., for military purposes – and what tools there are to prevent them.

What information did we gather and how did we adjust our project?

According to Prof. Nixdorff’s assessment, our project and its individual components do not harbor any intrinsic biosecurity risks. She is not aware of any examples in which B. subtilis have been used as bioweapons. Further, she stated that it is very challenging to make a non-pathogenic bacterium – such as the S1 organism B. subtilis – pathogenic by genetic modification.

Nevertheless, in order to obtain an additional safety level for the release of our sleeper cells, she suggested the use of so-called suicide genes. This encouraged us to design our kill-switch. In order to be able to carry out a proper risk management, she also suggested a risk analysis.

As a founding member of the interdisciplinary organization IANUS for peace research, Prof. Nixdorff emphasizes the inclusion of different interest groups in the research process. In doing so, she supports our initiative to make Responsible Research and Innovation (RRI) a central part of our Human Practices. In doing so, opinions and concerns of other scientists, of political institutions as well as the broader society will be considered.

Dr. Johannes Fritsch

Dr. Johannes Fritsch studied microbiology, biochemistry and molecular biology/genetics at Humboldt-Universität of Berlin and received his PhD in 2011 from the Institute of Biology at Humboldt-Universität and Penn State University (Pennsylvania, USA).

Since 2013, he has been a scientific officer in the presidential office of the National Academy of Sciences Leopoldina, where he is involved in topics such as personalized medicine, pandemic control, genome surgery and regulatory frameworks of genetic engineering, and the responsibility of science.

Since 2015, he has headed the office of the Joint Committee on the Handling of Security-Relevant Research of the Leopoldina and the DFG in Berlin.

Why did we talk to him?

We talked to him in order to evaluate the safety aspects of our project, determine possible sources for headwind from society and understand the precautions we would need to fulfill according to the law in case we want to actually realize our theoretical project.

What information did we gather and how did we adjust our project?

Based on his opinions and recommendations we decided to check and make sure that the target sequences of the phages are as precise as possible in order to avoid, that the phages do not only attack the pathogens, but also other cells.

Further, we decided to work on the acceptance of GMOs by constructing a survey that deals with the risks, prejudices and fears connected to GMOs, what people think about GMOs and synthetic biology in general as well as what they request from science communication.

Lastly, like he had recommended, we contacted the LAGeSo and the ZKBS to get more information about the precautions we would need to take and to what extend we would need to fulfill following collection of national laws and regulations (i.e., Gentechnikgesetz, Biowaffenkonvention,…) if we actually wanted to realize our theoretical project.

Federal ministry of environment, nature conservation and nuclear safety

We had the possibility to talk to a staff member of the federal ministry of environment, nature conservation and nuclear safety. Although the talk gave us a lot to think about, the person did not want to be officially mentioned nor should his words be published. The following is only a summary of the content of the talk and its key points.

Why did we talk to the federal ministry?

To receive an overview about the most important laws and regulations regarding the handling of GMO´s and consequences which could arise from various scenarios.

What information did we gather and how did we adjust our project?

The main message we took away from this interview is to balance the risk of a GMO release and it´s benefit. It is crucial to keep the consequences in mind which could happen and result in an ecological catastrophe. As well there was an ethical question on how much humans could or should be able to change in nature and how there should be a limit to ensure that we “don´t play God”. Thus, we decided to pay close attention to biosafety and reached out to different experts with whom we discussed methods on how to make our project safe.

Dr. Michael Vockenhuber

Dr. Michael Vockenhuber is the Biological Safety Officer at the Technical University of Darmstadt and thus our contact person for safety issues.

Why did we talk to him?

We talked to Michael Vockenhuber to get an overview about possible risks of GMOs, which also affect our project, and about ideas on how to increase safety.

What information did we gather and how did we adjust our project?

The biggest problem with the controlled release of GMOs are the strict regulations that apply to them in Germany and Hesse, because even the controlled release of GMOs can have unpredictable consequences that can affect the ecosystem. Instead, our biofilm should be used in a closed of optimally controllable system. This reinforces our idea to develop a kill-switch that prevents the unintentional release of GMOs from the biofilm.

Further, he pointed out that it must be ensured that the phages only attack the desired pathogens. Because phages are very specific, they only attack the targeted organism and no other bacteria present in the environment or in the biofilm itself. In addition, there is a risk that the metabolic load caused by the phages will become too great and the bacteria will no longer be able to perform their actual task.

Dr. Swantje Straßheim

Dr. Swantje Straßheim works in the secretariat, which gives scientific support to the Central Committee on Biological Safety (ZKBS).

The ZKBS is a voluntary committee that advises decision-makers in politics and administration through professional statements.

The main work of the ZKBS consists in subjecting organisms to a risk assessment or assigning genetic engineering work to a security level. They can also give suggestions for security measures if necessary.

Why did we talk to her?

We spoke to her to evaluate the safety aspects of our project with experts we talked to a representative of the ZKBS. As they are experts for GMOs, we were able to discuss the safety and the problems of GMOs in general, as well as our host organism B. subtilis. We could also get information about the legal difficulties in the release of GMOs. Those are very important aspects for our project.

What information did we gather and how did we adjust our project?

Since it is a lot safer to only release GMOs in a closed system, we continued to look into possible areas of application in which our GMO does not have to be released into the environment.

B. subtilis belongs to the category of microorganisms (MOs) that are “generally recognized as safe”. As long as no pathogenic factors are added to this MO, they are very safe to work with and a good choice for our project. A very important aspect that the ZKBS examines in a project is the retrievability of the GMO. We want to achieve this point through our implemented kill-switch.

Dr. Straßheim mentioned the problem that the term “synthetic biology” has a rather bad reputation in the general public. Reason for that is failed communication between scientist and the general public. This has confirmed us in our belief that we want to have a big focus on science communication in our project and include the opinions and fears of the public in our work.

Final statement

Over the course of our iGEM year we managed to reach a great number of experts from various fields and departments in order to optimize our project PHIRE BYRD and bring it closer to its actual realization and implementation.

We are immensely grateful to each and every one who has offered us their help.

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