“Science is well-ordered when the inquiries it pursues are those that accord with the agenda that would have been set by a group of discussants fully informed of the scientific opportunities, fully informed of one another's needs, and dedicated to doing the best they can to accommodate the needs of all” 
- Philip Stuart Kitcher, philosopher of science
The quote by Kitcher describes how science is serving its purpose when scientists prioritize inquiries that consider diverse perspectives, address shared needs, and aim for the collective benefit. Among multiple questions which the ideal of well-ordered science prompts us to consider, a fundamental one is about setting research priorities. Considering the vast array of potential topics and the inherent limitation of resources, what should scientists investigate, and how should the resources be distributed between different lines of investigation?
These questions remained important throughout our project and guided us on our way to ensure that our project is meaningful and beneficial for the world.
From the initial days of brainstorming, we welcomed input in the form of meetings, literature reviews, and reflections within the team. This allowed to decide on impactful and innovative aspects our project should contain. For us, the goal became contributing to foundational science in a way that has an immediate application beyond academia. Accordingly, when designing our project, we decided to address problems of high environmental and social relevance, such as enabling large-scale, continuous microbial bioproduction and sustainable vitamin B12 biosynthesis.
Consideration of the questions Kitcher posed requires a certain kind of epistemic humility: openly acknowledging that we might be wrong in what we consider a valuable contribution to science and society. Therefore, continuous engagement with trustworthy sources informed project development and was instrumental in evaluating whether we were addressing the prioritized problems in the most effective way.
INTEGRATED HUMAN PRACTICES
Here, you can follow our journey from the initial days of brainstorming the project idea to its final shape. You can read more on:
- individual meetings and in-person engagment with relevant stakeholders, experts and public (click on the “Additional meeting notes” button for more!)
- the reflective process based on meetings and literature reviews throughout the project in the “Reflection” sections
- a given meeting's influence on the project under “Integrated feedback”
Brainstorming with iGEM Freiburg 2022
University of Freiburg
January & February 2023
The majority of the iGEM Freiburg 2022 team members are our supervisors, many of them aspiring synthetic biologists, and the very foundation of our team: without them, we would not know of iGEM. For over a month, we met on a weekly basis to brainstorm and evaluate high-impact, innovative, and inspiring project ideas.
- Ask regional people, companies, and organizations about the problems they face and evaluate how synthetic biology could address them.
- Reach out to global organizations and think tanks to hear their suggestions on the most pressing problems that biologists could adress.
- Connect with research groups at the University of Freiburg and other universities whose work relates to the project ideas we consider.
- Get in touch with other iGEM teams that worked or are working on related project ideas to see if we can learn from them or collaborate with each other.
- Do an extensive, in-depth evaluation of the proposed project ideas, along with literature reviews on the latest developments in synthetic biology.
To gather inspiring and impactful project ideas, we followed iGEM Freiburg 2022 team member suggestion and reached out to various regional companies and organizations, including Fraunhofer Institute for Solar Energy Systems; dairy manufacturer Schwarzwaldmilch; plastic manufacturer DOMO, the major of Freiburg city, Freiburg University Hospital, Freiburg-based brewery Ganter, to global organizations including The Good Food Institute; Effective Thesis; New Harvest, ALLFED - Alliance to Feed the Earth in Disasters. We connected with researchers at the University of Freiburg, ETH Zürich, University of Tübingen, Reutlingen University, University of Jena, TU Munich and got in touch with other iGEMers from St Andrews iGEM 2012, iGEM TU-Braunschweig 2023, UCSC iGEM 2017. In parallel, we continuously reviewed the literature on the newest developments in the synthetic biology field and looked into cyanobacteria deployment specifically.
Reflection: the potential we see in cyanobacteria
Why did cyanobacteria catch our attention?
From an environmental standpoint, the use of cyanobacteria as more sustainable microbial biofactories holds immense potential since these organisms can efficiently convert atmospheric CO2 into valuable products, utilizing light as an energy source [2, 3]. Additionally, due to their ability to produce oxygen and sustain DNA damage from increased radiation , some cyanobacteria species can provide vital support for Mars exploration and eventual habitation . By further leveraging cyanobacteria's abilities, we can establish sustainable bioproduction systems for essential nutrients (like vitamin B12) which would both enhance the feasibility of long-duration space missions and boost sustainable bioproduction on Earth.
Clearly, these bacteria were worth further exploration, but none of us, nor our supervisors had previous experience working with thes organisms. Therefore, we reached out to cyanobacteria-focused research group leaders at the University of Freiburg.
- Consider the novel genetic circuit idea Prof. Wilde suggested for biocontainment: coupling the survival of bacteria to the presence of a particular compound.
- The bacteria would only survive as long as the specific degradable molecule is available.
- Explore the use of toxin-antitoxin systems to regulate cell growth and survival aswell as inducible promoters and aptamers to sense the compound, for implementation in the suggested autoregulatory system.
- Note that E. coli are more suitable for the initial testing of the system and cyanobacteria can be explored in parallel.
Prof. Dr. Annegret Wilde and Prof. Dr. Wolfgang R. Hess
University of Freiburg
At the University of Freiburg, Annegret Wilde and Wolfgang Hess are group leaders of the Wilde Lab and CyanoLab, respectively. Both focus on cyanobacteria microbiology, therefore they have in-depth expertise on the current and future deployment of cyanobacteria in research and industry.
Part of this conversation became the basis of our project and we remained in touch with both professors throughout our iGEM journey.
Inspired by the idea of coupling cell survival to the presence of (any) compound, we decided to look into the literature to explore current efforts in this direction. The idea of creating an autoregulatory system sounded intriguing and we started brainstorming possible applications of such a system.
Reflection: on choosing a project idea that combines foundational science with real-life applicability
We were intrigued by the idea of creating an autoregulatory system with a wide variety of potential applications, like the one Prof. Annegret Wilde proposed. Upon further literature review, we found no reports of such a universally applicable, dynamic control system design. Also the success of foundational advance-focused project of last year’s iGEM team from Freiburg inspired us to develop our project into foundational science direction. At the same time, we wanted to see its applicability beyond the lab bench. Question arose: can we combine both?
Since cyanobacteria remained of great interest, we considered making it a side branch of our project while we do the initial testing of the autoregulatory system design in a faster-growing organism, E. coli. Specifically, cyanobacteria were considered for sustainable vitamin B12 production and, once coupled with the autoregulatory system, could serve as the applied contribution of our project.
Keeping both the autoregulatory system idea and cyanobacteria as sustainable chassis exploration meant work had to be divided to cover both: therefore, we split into subgroups to research the single components for the project.
We also realized that for a project with a lasting impact, continuous engagement with experts and potential stakeholders is crucial: already the first meetings had enriched our perspectives on possibilities synthetic biology holds and shaped the direction project was taking.
Dr. David A. Russo
University of Jena
David Russo is an Alexander von Humboldt Postdoctoral research fellow at the University of Jena. Parts of his research include photosynthetic biotechnology. We got in touch with David Russo after listening to his engaging talk on the CyanoWorld YouTube channel which explored cyanobacterial secretion of proteins and small molecules for ecology and biotechnology. The initial conversations happened via email, contemplating various cyanobacteria-related project ideas, from omega-3 fatty acid production to living materials and biophotovoltaics. Finally, we met online to discuss the idea of the proposed autoregulatory system and its potential implementation in cyanobacteria. We opted to talk about the finetuning of the autoregulatory system elements (riboswitch and toxin-antitoxin system) for the deployment in cyanobacteria, as well as the use of Arthrospira platensis (spirulina) as a chassis for vitamin B12 production (given it has a more immediate application in the nutraceutical industry compared to lab model strains).
- Test vitamin B12 production with cyanobacteria model strains first, since Arthrospira platensis is difficult to transform
- For the toxin-antitoxin system deployment in cyanobacteria, explore a recent publication on a riboswitch-inducible CRISPR/Cas9 system in Synechocystis Sp. PCC 6803 that employed mazF 
Until the meeting with David Russo, we still considered A. platensis as a potential chassis for testing B12 production. Yet, given the challenging transformation David Russo mentioned, we decide to focus on one of the cyanobacteria model strains instead.
- Keep the system as simple as possible to minimize additional burden on cells. Therefore, the two-plasmid system is likely not desireable; this applies especially if the system is to be considered for application in large-scale fermentation processes.
- Make sure to characterize every single part sufficiently before combining them.
- Note that the greatest challenge will be to fine tune expression rates of toxin and antitoxin for the system to function properly.
- For bioproduction purposes, test and fine tune the final system in a bioreactor setting, because the bacteria are likely to behave differently there.
- Design the final system for genome integration. Selection utilizing antibiotic resistance is not feasible on larger scales.
Dr. Max Mundt
Max Mundt is a synthetic biology researcher turned founder turned investor. Our supervisors from the iGEM Freiburg 2022 team recommended getting in touch with him to assess the applicability and upscaling potential of our project for bioproduction purposes. One of our questions was the feasibility of expressing the envisioned autoregulatory system on one plasmids, instead of two, to improve its retention in cells.
Initially, the design we had considered was based on arranging the genes on two plasmids, which would be dependent on each other for the retention of the whole system. Considering the feedback we got, we decided to rethink this design to ultimately fit it on one plasmid. While we had only planned lab-scale experiments for the system, we now realized that the final testing in a bioreactor might be needed as well to assess its industrial potential.
Reflection: why engagement with industry is important
We noted Max Mundt’s suggestion to test our construct in a bioreactor setting and were interested in exploring the importance of this step through further literature research. Microbial bioproduction, the field we see as an immediate application of our autoregulatory system, suffers greatly from lab-to-bioreactor transition challenges . Industrial strain performance can not be extrapolated from lab-scale experiment unless factors like bioreactor-specific stresses (e.g. pressure, shear, pH), overexpression of production genes and prolonged cultivation times are taken into account [7, 8, 9]. For example, the same microbial population can consist of <3% non-productive cells at generation 37 and shift to 96% non-producers as it reaches generation 60, which corresponds to the time required to grow a sufficient culture in a small bioreactor for scale-up .
The apparent gap between lab and industry means that potentially impactful synthetic biology innovations remain in their nascent state . Therefore, decisions were made to engage with strain optimization and industrial bioproduction experts at enGENES Biotech and BRAIN Biotech (see meeting notes below) to increase the applicability of the autoregulatory system we are creating, while also noting potential improvements.
Santa Cruz iGEM team 2017
McKenna was a member of the University of California, Santa Cruz iGEM team 2017 and wrote her master’s thesis on the basis of this project . The iGEM project and part of the thesis proposed a way to modify the cyanobacteria strain S. elongatus PCC 7942 for vitamin B12 production, an intriguing suggestion that we wanted to test in practice. We were glad to have a meeting with McKenna since her master’s thesis was the only publication we found on S. elongatus PCC 7942 modification for B12 production.
- Build upon Hicks’s master’s theses to further test the cyanobacteria model strain S. elongatus PCC 7942 usability as a novel B12 biosynthesis chassis
- Consider further testing in A. platensis, a cyanobacteria strain that is approved for consumption and hence would be an even better fit for B12 production
- Note that extraction and quantification of vitamin B12 is challenging
- Make sure that the origin of replication of our plasmid is compatible with S. elongatus PCC 7942 for future implementation.
Before, we considered a couple of cyanobacteria model strains for B12 production and chose to work with S. elongatus PCC 7942 given Hicks mention of better data availability. Following her advice to look for the origin of replication compatibility, we selected a shuttle vector designed specifically for use in S. elongatus PCC 7942 (developed by iGEM Marburg 2019 team). Lastly, we employed the 2 genes, ssuE and bluB, for B12 de novo biosynthesis based on the theory Hicks’s developed in her thesis.
- MazF, the toxin we utilize in E. coli, likely does not mediate cell-death but only growth inhibition in cyanobacteria. Still, this should be sufficient for CELLECT to work in cyanobacteria since unproductive cells would be at a comparative disadvantage compared to the productive ones.
- A different riboswitch is needed since the one we implement in E. coli is derived from the very same organism and is triggered by pseudocobalamin as well. This would be the direct precursor which we expect to be produced natively in S. elongatus PCC 7942, the strain we would want to work with. Hence, this structure is not ideal as we want the riboswitch to respond exclusively to B12.
- If we design the system in E. coli first, we should be aware that it will have to be modified again before it can be transferred into cyanobacteria, because it will function differently there.
- The advantages of using cyanobacteria as a host-organism for bioproduction processes are simplicity and inexpensive cultivation. For example, when used for nutrient supplementation, purification might not be needed as many cyanobacteria strains are edible, the most well-known example being A. platensis or spirulina. Yet, food regulations need to be considered. Consumption is approved only for specific strains, and in addition to this, we would develop genetically modified organisms (GMOs), which, especially within the European Union, are strictly regulated.
Prof. Dr. Annegret Wilde and Prof. Dr. Wolfgang R. Hess
University of Freiburg
Almost two months of research and project development had passed after the first meeting which inspired our team to create the autoregulatory system. Now, we met again with Annegret Wilde and Wolfgang Hess to discuss the latest ideas, specifically focusing on the CELLECT implementation in cyanobacteria.
From the meeting we learned that our initial idea of using the E. coli riboswitch in the cyanobacteria would likely not be successful due to its possible activation by natively-produced pseudocobalamin. Therefore, to adjust the autoregulatory system to cyanobacteria, we decided to test another, B12 specific, riboswitch derived from P. freudenreichii. This riboswitch is supposedly not activated by pseudocobalamin and hence suits our objective to detect exclusively B12.
Reflection: combining it all
Engaging with experts from industry and academia to validate and improve the concept of our project was the next important step for the development of our idea towards real-world applicability.
Consultation with Max Mundt, someone with expertise in the biotechnology industry, allowed us to consider adjustments in system design that would be necessary for application in large-scale fermentation processes. David Russo’s advice drove us to carry out our experiments in relatively well researched model organisms. This matches the approach of McKenna Hicks, who started with B12 production in S. elongatus PCC 7942 despite her ultimate goal to establish B12 production in A. platensis. Direct exchange with her gave us valuable insights into potential challenges we could be facing along the way, learning from her experience.
The discussion with Annegret Wilde and Wolfgang Hess revolved around the functionality of specific system parts in cyanobacteria, as they would likely work differently compared to E. coli. The points they made outlined the adjustment that would be necessary to implement our system into different organisms. Depending on the synthesis or degradation pathway, the riboswitch and the toxin/antitoxin system might need adjustment according to respective conditions. Going back to the transfer from E. coli to cyanobacteria, in our case, a different riboswitch than the one we utilize in E. coli is required because of different affinities to B12 precursors. Additionally, the ratio of toxin and antitoxin expression would need to be adjusted because of reduced impact of the toxin on cyanobacteria.
To us, this emphasizes the relevance of a considerate project development process, striving to achieve excellent functionality of an individually designed system according to the desired application.
Dr. Christian Fleck
University of Freiburg
Further optimization of the autoregulatory system would require mathematical modeling, yet we did not have anyone with sufficient expertise among our team members. Here, Christian Fleck was the right person to reach out for two reasons: besides being head of the Spatial Systems Biology Group at the University of Freiburg Centre for Data Analysis and Modelling, he was also PI for iGEM gold-medal winning teams at the University of Wageningen. As none of our team members had previous experience working in research nor taking part in a competition such as iGEM, we considered it beneficial to hear how other successful iGEM teams went about reaching their goals. Therefore, we appreciated the opportunity to talk to Fleck about everything from modeling to bringing great iGEM project ideas into reality.
- Ensure project feasibility and relevance, with a focus on broad ideas that are relatable to a wider audience.
- Explore and leverage the strengths of individual team members, supervisors, and the university research groups to create a realizable, high-impact project
- Establish a clear team structure with assigned roles and specialists for different aspects (e.g. human practices, finances). Division of responsibilities reduces confusion, fostering a more efficient work environment.
Following the meeting, we welcomed a doctoral student from Fleck’s group, Jonas Pleyer, as a team supervisor and mathematical modeling tutor: that allowed us to learn modeling basics and improve our system.
Given Fleck’s emphasis on having a manageable and feasible project concept, we arranged further meetings with experts from industry and academia to validate different parts of the autoregulatory system.
- Stay with S. elongatus PCC 7942 as our model strain since it is easy to work with; naturally competent and relatively fast-growing. S. elongatus UTEX 2973 grows twice as fast as S. elongatus PCC 7942 but the rapid growth comes with high light intensity requirements and consequently higher energy expenditure and excess heat generation (both of which we would like to avoid).
- Start with testing B12 production in cyanobacteria which in itself is a great project. Only once that is working, proceed with the autoregulatory system implementation.
- Consider the future of the project: given the photosynthetic properties of cyanobacteria, a sustainable scale-up of bioproduction could include a big fermentor on a rooftop where sunlight is utilized as an energy source.
University of Freiburg
Khaled Selim is a research group leader, focusing on cyanobacteria microbiology. We were in touch during the early months of iGEM regarding various cyanobacteria-related project ideas and we met in person shortly after Selim moved his lab to Freiburg. We were interested to hear his opinion on the autoregulatory system design early on and how realizable its implementation in cyanobacteria for enhanced B12 production would be. Additionally, given Selim’s experience with S. elongatus PCC 7942, the strain we considered working with, we wanted to learn more about its cultivation specifics and discuss the potential use of S. elongatus UTEX 2973- a fast-growing strain that e.g. iGEM Marburg 2019 team worked with as well.
Inspired by iGEM Marburg 2019, we considered the possibility of using fast growing strains, like S. elongatus UTEX 2973, for our project. However, based on the feedback from Khaled Selim, we decided to stay with the model strain S. elongatus PCC 7942. Instead of trying to test the autoregulatory system and B12 production in parallel, we followed his suggestion to start with the latter first, and only once successful, try integrating the whole system.
Dr. Juergen Mairhofer and the team
enGenes is a biotechnology company based in Vienna, Austria, offering the development of customized solutions for the cost-effective production of recombinant proteins in microbial expression systems. As our system was potentially applicable in large-scale microbial bioproduction, we seeked advice from the biotechnology company regarding its implementation and usefulness in this domain.
- Ensurance of genetic stability by use of antibiotic resistance genes is not sustainable on a larger scale, considering cost of the antibiotics and the necessity for waste-water treatment. Instead, integration of the additional genes into the genome of bacteria is a common practice in bioproduction.
- Test the single parts individually over a longer time span. Precise timing is required for proper functionality of the system. Assemble it step by step.
- The greatest challenge ahead: stabilization of the toxin gene, as there probably is a high selective pressure against maintaining its function. Cells with no/little toxin expression would likely take over the culture because of their evolutionary advantage compared to the cells containing the functioning system.
We put emphasis on the advice from enGenes to characterize all individual parts first before combining them. Moreover, we noted the genome integration for later attuning of the system to accommodate microbial bioproduction requirements.
Reflection: on phenotypic instability and CELLECT application
We were well aware of the term “genetic instability”, exemplified by e.g. plasmid loss. There is a more intricate side to it though, which does not involve the loss of genes but still results in the loss-of-function: phenotypic instability. As mentioned by enGENES Biotech, genome integration is a common practice in bioproduction to secure a construct retention. However, no satisfying solutions have been developed so far to ensure persistent gene expression [8, 9]. Hence, the productivity and stability of industrial strains remain severely limited. Currently available options, like growth-coupled production, require genome in silico modeling , while multi-layer genetic circuits rely on specific regulatory components and tailored mathematical models to optimize the circuit´s design ; both of which are labor-intensive tasks. The challenge remains to create an optimal gene expression control system that can be easily adjusted to various compounds and host organisms, one that is able to integrate multiple signals in a timely manner, assure genetic stability and minimize the metabolic burden . Does CELLECT’s versatile design make it a feasible solution to phenotypic instability? Potentially, as enGENES agreed. Yet, besides ensuring the robustness of the individual CELLECT parts, we have to test the metabolic burden it imposes on the cell and how that impacts the production yield. For that, precise quantification of the compound we produce, vitamin B12, is required.
- Change the ethanolamine medium testing strategy: instead of transferring E.coli from LB medium to ethanolamine medium, let E.coli grow in B12-supplemented ethanolamine medium before the assay (less stress for cells; shorter lag phase due to adapted metabolism)
- Always use the same E.coli clone for the assay as the maximum growth can vary greatly
- Consider changing the carbon source in the ethanolamine medium from glycerol to ethanol since ethanol is also a byproduct of ethanolamine breakdown
- Continue developing the ethanolamine medium assay: the idea is interesting and it would be a valuable, easy-to-use detection tool
Prof. Dr. Matthias Boll
University of Freiburg
Matthias Boll is microbiology professor at the University of Freiburg and Boll's research group leader, with a focus on the metabolic biochemistry of microorganisms. We reached out to him due to issues with an alternative vitamin B12 detection method (ethanolamine medium assay) we were trying to establish to assess its general usefulness and potential improvements.
Reflection: the challenges with vitamin B12 detection
There is an abundance of methods available for the detection of B12, yet most of them are limited in access due to the requirements for large and expensive equipment, complicated reagents, long assay times, low tolerance to complex matrices, poor limits of detection or insufficient specificity to distinguish between the different forms of B12 . To establish a detection method that is suited to our project’s requirements, we first tested the Demeditec B12-ELISA kit for foods, which turned out not to work well for our application. We ended up testing two separate detection methods from one paper, a riboswitch sensor and an ethanolamine utilization assay for E. coli . While we got mixed results when trying to establish the riboswitch sensor, the ethanolamine assay looked promising (Results/Sensing B12), at least for qualitative detection of B12. We therefore developed and tested it for quantitative measurements, seeking and implementing the advice of Matthias Boll in the process. It evolved to the point where we found it to be a valuable detection method to optimize not only for our purpose, but for other researchers to implement in their work. This assay is affordable and simple: it consists of growing E. coli in a specific medium, in which the cell growth is dependent on the presence of B12. Yet, for the autoregulatory system testing while developing this assay and also for its later accuracy assessment, we still needed a reliable, quantitative method for B12 detection. Therefore, we contacted Luciana Hannibal, a researcher at the University Medical Center Freiburg, specialized in B12 metabolism.
Freiburg Center for Pediatrics and
Luciana Hannibal is Head of the Metabolomics Core Facility at University Medical Center Freiburg, studying molecular mechanisms of disease and new therapeutic targets with focus on sulfur-containing metabolites and Vitamin B12. We got into contact with her because we were looking for a detection method to quantify B12 production.
- We should be able to convert most of the different B12 forms inside the samples to hydroxocobalamin (OHCbl) by exposing them to white light for about 2 hours.
- Look at the research that is being done at Quadram Institute (United Kingdom). They work on improving national food stability, with a lot being done in regards to B12. Prof. Martin Warren’s work could prove to be especially beneficial for us as he studies B12 metabolism in bacteria.
- Commercially available B12 for supplementation is almost exclusively produced in China. Therefore, there is a geopolitical interest in the establishment of large-scale B12 production in Western countries.
- The most simple and inexpensive way to obtain highly pure B12 is to treat all B12 forms with cyanide for conversion to cyanocobalamin (CNCbl) with cyanide. It is the most stable cobalamin, preferred for distribution in supplements.
Hannibal confirmed that we could convert the B12 in given samples to OHCbl by way of deliberate exposure to white light. Therefore, we always included a step of deliberate white light exposure for a time period of 2-3 hours during sample preparation for LC-MS.
- People show great interest in exploring and understanding foundational synthetic biology principles, such as “How bacterial DNA is changed?”. Explaining these principles to them can serve as a foundation for further engagement and interest in synthetic biology
- Explaining our project, the autoregulatory system, taught us how to present and explain our system to people with different degrees of expertise
CIBSS Open Door Day
Centre for Integrative Biological Signaling Studies
The CIBSS represents one out of two Clusters of Excellence associated with the University of Freiburg and sets the foundation for Freiburg’s iGEM teams. The annual Open Door Day takes place to make the work done at CIBSS accessible to anyone who is interested, communicating the meaning of the research done to the general public. For us, it was an opportunity to present our project in front of a small audience and talk to interested citizens afterwards (Education).
Reflection: science literacy and what we did about it
In the introduction, we touched upon Philip Kitcher’s view of well-ordered science which conceives “scientists [..] as artisans working for the public good” , a view we embrace. Yet, an important question remains: how does the public know that we do? Why would anyone beyond the lab doors trust that we are putting the resources into good use? As historian of science Naomi Oreskes writes, trust is an important part of effective science communication- and one way to create it is by building connections from the research institutions into the local communities . CIBSS Open Door day surely allowed us to connect with the neighborhood, but it also highlighted the importance of science literacy. Why? Because it empowers people to have a closer engagement with the newest developments in fields like synthetic biology and consequently provides bases for a better dialogue with scientists; one which is enriching for both sides. Already during the Open Door days, we observed keen interest from visitors to learn about foundational synthetic biology principles. Making biology more accessible in an engaging way became an inspiration for us, encouraging more education-related projects to come (Education).
Biology Faculty Day
University of Freiburg
The Biology Faculty Day at University of Freiburg offers the chance for students and researchers at the Faculty for Biology to get to know their peers and the work that is being done at the faculty. It is a celebration and appreciation of biological sciences and scientists. We presented our project in front of an audience of about 100 students, researchers and professors to obtain feedback on our ideas.
- Prof. Barbara Di Ventura: The toxin we implement is likely to be subject to loss-of-function mutation, because of the high selective pressure against its function. Bacteria that inherit a mutated toxin gene would then be able to take over the culture due to the gained evolutionary advantage over cells that possess a functional toxin gene. This could be prevented by creating an overlap of the toxin gene and a selection marker gene on a secondary reading frame.
- Prof. Annegret Wilde: The impact of potential toxin mutation in some cells could be mitigated by inducing the system in the stationary growth phase of the bacteria. Therefore, the mutation could not spread across the culture.
We learned that the risk of the mutation of the toxin could be a severe hindrance for the success of our project and came up with a second design of our system, which would allow us to address the instability of the toxin (Design).
- Loss of toxin expression is more likely to occur due to mutations in the promoter region regulating the toxin gene and not in the toxin gene itself. Our idea to implement the selection marker secure promoter function makes sense
- There is more selective pressure against additional protein expression of any kind in E. coli than in cyanobacteria as the latter generally are less energy-limited
Wolfgang R. Hess
University of Freiburg
Wolfgang Hess is the leader of the CyanoLab at the University of Freiburg, with main focus on cyanobacteria microbiology. After the feedback received at the Biology Faculty Day, we met with him to discuss potential solutions to the potential for toxin mutation that was suggested then.
Following Hess’s advice to focus on securing the promoter, which was suggested to be the part most prone to mutations, we came up with an idea to employ a selection marker gene downstream of the promoter, which would only be expressed if the promoter was working. As Hess confirmed, this re-design of the system could work to limit the risk of toxin loss-of-function. Therefore, we continued with the cloning of this construct.
Dr. Janina Kölschbach, Jens Peter Gersbach, Jan-Eike Domeyer
BRAIN Biotech is a company based in Zwingenberg, Germany, with focus on research and development for customized enzymes, optimized microbial strain development and efficient bioprocesses. We talked to B.R.A.I.N. to hear first hand from stakeholders how feasible and applicable CELLECT is at its current state.
- Integrate the system into the genome for application in industry.
- Secure the TetR promoter as it is the part of our system most prone to mutation. Since we carry out the production in M9 medium, we might consider adding a crucial part of a metabolite pathway, e.g. for an amino acid, between the promoter and the riboswitch upstream of the toxin gene to secure promoter functionality
- Cells might lyse randomly, also productive ones, which would lead to release of B12 into the medium. Unproductive cells could pick up this released B12 to not be affected by CELLECT. Lysis rate depends on the organism and the compound produced. For the proof-of-principle, this should not be a concern, although for actual application we might have to explore alternative solutions and/or other organisms
- Consider replacing the chemically induced promoter TetR to a heat-induced promoter which would make CELLECT more feasible for use in a bioreactor
- Start looking into patent law to check whether CELLECT includes any parts that are the properties of companies and to ensure that our designed plasmid is non existing in any kind of registry. It is important in case we want to further develop CELLECT to make it market-ready
The B.R.A.I.N. feedback confirmed our approach of securing the promoter controlling toxin expression. Additionally, following up on their suggestion, we collected and sent for LC-MS measurements supernatant samples from the production cultures to determine how much B12 is released by lysing cells.
Reflection: on the good, the bad and the future
Presenting CELLECT to various people from academia and industry allowed us to gather perspectives on both its potential and shortcomings. The most notable advantage remained the versatile, comparatively simple design of the system, with minimal mathematical modeling requirements and applicability across different fields. Among the needed improvements was stabilization of the most vulnerable part of CELLECT, the toxin gene and its promoter, to minimize the occurrence of mutations. In response, we developed new versions of CELLECT, which, while not tested during this project due to time constraints, encompass toxin-stabilizing elements. From the industry meetings, we noted that an industrially-applicable version of the system would also need to be genome-integrated to avoid antibiotic-based selection and the financial and environmental burden it implies on large scale. Lastly, replacement of chemically inducible promoters with constant or heat-inducible ones would make CELLECT even more applicable for bioproduction and degradation purposes. Nearing the end of the project, we came across a recent publication on a novel multi-control system which employed a mechanism similar to one we propose. Utilizing ligand-dependent riboswitch, tightly controlled inducible promoter and low-copy number strain, the system is designed to enable cloning of toxic or unstable genes . The study supports some of the observations during our project (such as low toxicity of MazF toxin in E.coli strain harboring native antitoxin) and serves as an intriguing outlook on yet another potential application for CELLECT.
- Over 90% of the commercially available B12 is produced in China
- Bioproduction of B12 currently results in a lot of cobalt waste
- There is a shortage in therapeutic B12
- Supplementation with high orally administered doses of B12 might decrease absorption in humans because of changed proportions between B12 producing and B12 consuming bacteria populations in the gut microbiome
- Hydroxocobalamin (OHCbl) is the preferred form of the vitamin for therapeutic injections to treat severe B12 deficiency
- Most B12 supplements contain cyanocobalamin (CNCbl), because this is the B12 form that is the cheapest and most straightforward to produce. From there, other forms would be made by chemical synthesis
- Substances produced with and extracted from genetically engineered organisms do not fall under EU GMO-regulations
- From November 2023, only vegetarian food will be served at children day-care centers and elementary schools in Freiburg, a potential forerunner for all of Baden-Württemberg. This underlines the rise in popularity of vegetarian and vegan diets. Supplementation with sufficient amounts of B12 therefore becomes increasingly important, especially for children
Freiburg Center for Pediatrics and
Luciana Hannibal is Head of the Metabolomics Core Facility at University Medical Center, studying molecular mechanisms of disease and new therapeutic targets with focus on sulfur-containing metabolites and vitamin B12. As the project had reached the final phase, we met again with her and her Lab’s Lead Scientist Victoria Wingert to present what we have been working on. Moreover, we wanted to talk more about the relevance of vitamin B12 today, as the first meeting had given only a brief insight into the vast amount of information and expertise Hannibal had on this topic.
Reflection: importance of vitamin B12, from health to bioproduction
Only microorganisms can synthesize vitamin B12 by themselves. Other animals, including mammals, cannot, yet they depend on it. Consequently, sufficient availability of B12 is a basic requirement for human health.
Although there are certain gut microbiota that can produce the compound, B12 from these bacteria is not bioavailable to humans, meaning that sufficient amounts of the vitamin derived from nutrition can only be achieved by consumption of animal foods. It is worth mentioning however that even in farm animals B12 levels have dropped over the last 30 years. Many times, these animals are injected with essential micronutrients themselves as they do not get them from the food they are provided with .
Independent of that, plant-based diets gain more and more popularity. While this shift can eventually alleviate the threat to the environment that factory farming is, it could cause another basic concern for human health instead. Sufficient amounts of an essential micronutrient might not be guaranteed, with potentially increasing demand for B12 to the point where there is more demand than supply. Exemplary for the shift in this direction, children day-care centers and elementary schools in Freiburg switch to an exclusively vegetarian meal-plan with the start of the new semester, which is observed as a potential example for the entirety of Baden-Württemberg to follow .
All of this speaks to the fact that the availability of B12 in the present and in the future needs to be addressed. As chemical synthesis of B12 is extraordinarily ineffective, commercial B12 is commonly produced through large-scale fermentation using the microorganisms Propionobacterium freudenreichii or Pseudomonas denitrificans [18, 19].
The cobalamin forms extracted from the bacteria are then treated with cyanide for conversion to cyanocobalamin (CNCbl). If needed, conversion to the desired B12-form is accomplished by way of chemical synthesis. This could be done to, e.g., produce hydroxocobalamin (OHCbl) for therapeutic use, as this is the preferred version of B12 for therapeutic injections, or for production of OHCbl for cyanide detoxification .
The majority of B12 on the market is synthesized in China . This opens up a geopolitical conversation. With a B12 world market share of about 90%, the distribution of B12 around the globe is highly dependent on this one country. Establishment of B12 production outside of China would greatly benefit the worldwide distribution of power over this aspect of human health.
Another point is waste. The free cobalt that is required in culture media as a basis for the bacteria to produce the vitamin is never converted to B12 efficiently, causing a lot of cobalt waste that eventually ends up in the environment. Bioproduction in many western countries is subject to more strict regulations, implying the reduction of such waste. For example, researchers around Prof. Martin Warren at Quadram Institute (UK) do already confront this challenge by creating a sustainable bioproduction method of B12 in E. coli .
Martin Warren’s research group furthermore explores the influence of B12 on the gut microbiome. This falls in line with an emerging interest in the effect that the vitamin has on bacteria in the gastrointestinal (GI) tract. An exemplary cascade of action, explained to us by Luciana Hannibal, would be this: The gut microbiome of Inflammatory Bowel Disease (IBD) patients may inherit a comparatively higher population of microbes that consume vitamin B12 to produce B12 analogs in turn. On one hand, more B12 entering the GI tract is taken up by these bacteria, leaving less of it to be absorbed by the patient. On the other hand, those microbiota can produce additional B12 analogs, which are taken up into the bloodstream as well. The B12 analogs then compete with B12, meaning they bind to the enzymes to which the B12 molecules would normally bind, but do not help catalyze the corresponding reaction. This thereby causes insensitivity to B12 in the respective human. Moreover, oral supplementation with high doses of B12 might mitigate the shift towards higher B12 consuming microbe populations, thereby increasing the risk for B12 deficiency once lower doses of the vitamin are administered .
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