Proof of Concept
Lab Work +
Dry Lab +
Dry Lab +
Wet Lab +
Wet Lab +
Human Practices +
Human Practices +
Education and Communication +
Education and Communication +
During all phases of our project we reached out to different stakeholders who are either affected by our work or could help improve our iGEM project by providing valuable knowledge. Their feedback was an integral part of ensuring our project was scientifically feasible and had a positive impact on the world. Most of the time we received very positive feedback, but much more important was modifying our project reacting to concerns raised by these stakeholders. With their feedback they very much became a part of our project and we hope that our project will become part of their life too.
Project finding phase
Starting an iGEM project can be difficult. There are so many possible project ideas out there.
How should one be able to choose the best one of them?
Our vague initial idea was to use green organisms to produce - something.
But why? -
To be independent of fossil energy.
This was for sure the starting point of our project leading to an elaborate project idea.
So first things first. Which organisms should we use for our project?
Green organisms are considered to be more complicated to work with compared to
and no one in our team had experience with neither plants nor cyanobacteria. But from what we have overheard, using cyanobacteria might be the way to go, considering the short timeframe of our project.
Getting expertise on cyanobacterias
We were novices in using cyanobacteria. So the logical thing for us to do was to get in touch with people who know how to work with cyanobacteria.
iGEM Marburg Alumni
Previous iGEM Marburg teams had accumulated a lot of knowledge regarding cyanobacteria. Since we were a bit overwhelmed on how to start with our project we hoped to get some advice on which bacteria and equipment to use. The iGEM Marburg team shared their experience with different cyanobacteria strains and cultivation methods with us. Furthermore, they offered us to use parts and strains from their lab. Additionally we talked about which necessary equipment we needed to obtain for cultivating cyanobacteria (like shaking light incubators and light bulbs) and how to fund it (sponsoring, DIY).
Going into this conversation with a lot of uncertainty, all our doubts had vanished and we were confident that we would be able to work with cyanobacterias.
Now the next question was, how can we use cyanobacteria in our lab, which was not equipped for this task or can we find an alternative place for incubating cyanobacteria.
Where do we get equipment?
Dr. Nils Wieczorek is a team leader of the Bioeconomy and Bioresources at the University of Technology in Hamburg. His research addresses questions related to microalgae-based biorefinery as well as aerobic and anaerobic processes and natural products from residual materials.
Dr. Wieczorek gave us useful tips on how to work with cyanobacterial cultivation systems. We realised that obtaining hardware for ourselves would be quite expensive and were glad that he could refer us to the local research groups of Dieter Hanelt at the University of Hamburg, who might be able to help us out with potential lab space, lab equipment, ideas and their expertise.
And this is how we found a research group at our university where we can do our project without needing to obtain a lot of necessary equipment.
1st project idea - using thylakoid systems and modified photosystems to produce H
Now we could really start bringing forward our ideas on what to do with these cyanobacteria. Initially our research idea focussed on modifying the photosystem to enable H
production and isolate the thylakoid membrane for
But we did not know if this thylakoid extraction and handling would be possible for us to do in our lab. Can we use isolated thylakoid systems in our project?
Dr. Tarryn Miller is a biochemist at the research group of Prof. Dr. Tobias J. Erb at the Max Planck Institute for Terrestrial Microbiology in Marburg.
We seeked Tarryn Miller for her expertise on working with isolated thylakoids. According to her, isolated thylakoids can have long-term advantages compared to working
mainly because not the whole organism is needed. But the thylakoid system is fragile and the stability depends on temperature and light intensity. Further ideas to combine thylakoid extraction with microfluidics pose additional challenges, since we would also have to establish this method in our group from scratch.
We were torn back and forth since Tarryn Miller really had emphasised the advantages of a thylakoid system, but we could also see the challenges associated with this system. The good thing was that we got additional advice leading to a final decision.
Thylakoid systems are not ideal to produce H
Prof. Dr. Dieter Hanelt is a leader of a research group at the University of Hamburg at the department of biology. His research group investigates ecophysiology in aquatic systems such as the production of microalgae in photobioreactors through the usage of carbon dioxide from flue gas.
Dieter Hanelt first questioned the usefulness of using cyanobacterial thylakoids in our project and proposed that it might be easier to work with more stable chloroplast of plants like spinach or chlamydomonas since the stability of the thylakoid membranes is a problem. Secondly he doubted that it would be economically beneficial on an industrial scale to use this system to produce H
, since other methods like electrolysis are very cheap. Only if biomass is also used (e.g. for fatty acid production) microorganisms might pose an alternative strategy of producing H
We therefore focussed our research on finding interesting compounds which could be additionally produced by the cyanobacteria.
The creation of photosystem knockout strain will not be feasible
Dr. Kirstin Gutekunst from the Christian-Albrechts University Kiel works on the central carbon metabolism in cyanobacteria and plants as well as hydrogen metabolism in cyanobacteria.
Recent work from Kirstin Gutekunst has strongly inspired our project. By fusing the hydrogenase HoxYH to the PsaD subunit of photosystem I (PSI) direct electron transfer from the PSI to the hydrogenase is possible bypassing other competing metabolic processes which rely on the energy provided by the photosystem. This enables the photosynthetic production of dihydrogen from only light and water. We wanted to express similar fusion constructs and isolate the thylakoids to do
production of H
. A fruitful conversation with Kirstin Gutekunst let us realise that the creation of the necessary photosystem I knockout mutants was very difficult and took her group years to achieve, more time than that was available during our iGEM project. Therefore we looked into finding alternatives omitting the necessity of creating the knockout mutants like using RNA interference or CRISPR-based methods.
Biosafety and Security analysis stops our project idea
Before starting to work in the lab we had to get permission from the university and identify any dangers associated with the project to implement necessary safety measures to prevent harming us, others and the environment. With experts from the universities we identified risks associated with our project. While there were no biosecurity concerns we identified biosafety issues. The main dangers identified during this process are connected to managing the release of produced H
. As it turned out even the expected minimal amounts of H
we would have to deal with were considered dangerous and constant supervision of our lab work by an experienced researcher would have been necessary. This was not a possibility to accomplish because due to corona restrictions the amount of people working at the university was minimised and thus supervision could not be guaranteed by our PI.
With a heavy heart we had to decide to stop this project idea at this point since there was no way we could carry on with it. We had to look for an alternative project idea.
2nd project idea - CYP P450/reductase fusion proteins for terpenoid production
Based on our research carried out while finding alternative usage of the biomass as a byproduct of H
production, we bumped into the interesting compound class of terpenoids. These terpenoids have a broad field of application. They are e.g. drugs. The idea was to produce drugs like artemisinin with microorganisms, environmentally friendly and maybe cheaper than chemical alternatives was enticing, making them available in low-income countries at price rates people can pay arousing. Before determining a target product we looked for confirmation that our general idea can be done.
Deciding on a terpenoid part #1
Prof. Dr. Sascha Beutel is group leader at the Leibniz University in Hannover in the department of technical chemistry. His research focus is set on bioprocess engineering, bioanalysis, downstream processing, and the implementation of interactive, digitally supported laboratory infrastructure.
We explained our idea of using Cytochrome P450 (CYP) enzymes/NADPH-cytochrome reductase fusion proteins to increase the reactivity to Sascha. He seemed very excited about our project and similar fusion proteins have been expressed in
previously. He urged us to keep in mind that the expression of soluble and functional CYP enzymes is complicated, making codon-optimisation and using solubility tags necessary. Additionally, we should also look into NADPH regeneration systems. Finally, we realised that diversification of our terpenoid portfolio would be beneficial to prevent failing of the project if e.g. the expression of specific cytochrome P450 enzymes would not be possible. But which terpenoids should we focus on? From the beginning on we pursued artemisinic acid, which has gained a lot of research focus in the last years. So would it be the perfect compound for us?
Deciding on a terpenoid part #2
Prof. Dr. Christian B.W. Stark is a leader of a research group at the University of Hamburg and working on different aspects of modern catalysis and synthetic organic chemistry with starting materials from simple organic molecules to complex renewables (such as terpenes, fatty acids, and carbohydrates) and densely functionalized natural products. According to Christian Stark “that ship has sailed” and we needed another “banger alternative”. He advised us to work with terpenes which have known specific biological effects and properties, and are additionally already used in industry or research. He proposed producing the natural product paclitaxel, a chemotherapeutic for different kinds of cancers which naturally occurs in Taxus brevifolia. So we looked in the biosynthesis of paclitaxel but soon realised that it would be very complicated and likely impossible in the timeframe of an iGEM project to implement this in
Besides drugs and flavours, fragrances might also be interesting places to look for target molecules.
We then based our consideration of choosing the terpenoids we wanted to produce during our project on this advice. The optimal terpenoid should a) have a known biosynthetic pathway, b) be easily producible in our bacteria, c) have a useful function (drug, flavour, etc.), d) have industrial significance and e) cost intensive production so that microbial fermentation is competitive. Another aspect of our decision was based on the availability of plasmid and resources provided by our collaborators. Additionally, we wanted at least partly to preserve our initial project idea to use cyanobacteria as a source of green energy. The terpenoids we accordingly chose to produce are parthenolide, artemisinic acid, humulene and bisabolene.
Project planning phase
The next step on our journey was to address concerns raised during our project finding phase. The first problem we needed to solve was to express functional cytochrome P450 enzymes.
Expressing cytochrome P450 enzymes
Prof. Dr. Vlada B. Urlacher is research group leader at the Heinrich Heine University in Dusseldorf at the department of biochemistry. The characterization of new enzymes, enzyme engineering and immobilization, multi-enzyme cascades and biocatalysis as well as biotransformation are research topics she is focusing on.
As it turns out it is possible to apply some basic design considerations to increase the chance of success. The most important aspect would be to remove the membrane domains from the coding sequence. Optimally this is achieved by creating a library of different modified CYPs and reductase of distinct membrane domain length and screening them for their activity. Unfortunately, it seems that not all CYPs can be expressed and there is no way of knowing which CYPs cannot before trying it out. Additionally, His-tags are useful for isolation and detection of the enzyme. Another factor is that protein expression rates are very low and proteins are often not visible on SDS PAGE gels. Vlada Urlacher also recommended using the ATR2 reductase which seemed to work well for different CYPs.
This conversion led to the redesign of our constructs since we had not previously removed the membrane domains. We considered creating protein libraries but decided against it since we did not have the resources and time to screen all constructs. In conclusion we saw our strategy to diversify our project to increase the prospect of success confirmed.
We also needed to find a way to deal with differences between
and cyanobacteria since we wanted to produce some of our products in cyanobacteria as well as in
Designing constructs for
is much easier compared to cyanobacteria, since there is a vast number of well characterised parts like promoters or terminators, available from the iGEM registry. But for cyanobacteria (specifically
) this knowledge is much more limited and we needed help to find the best way to design our constructs for cyanobacteria.
Improving our cyanobacteria project
Prof. Dr. Ilka Maria Axmann is a group leader at the Heinrich Heine University in Düsseldorf in the department of biology. She has set her focus on extracellular matrix of cyanobacteria, bioreactor co-cultivation of heterotrophic and phototrophic microorganisms, synthetic gene regulatory systems in cyanobacteria and the circadian clock. Recent work of Prof. Ilka Axmann expanded the knowledge of different promoters used in cyanobacteria. She also advised us to design modular constructs which can be used in both organisms. There are parts available that we could use that are compatible with MoClo and can be very easily combined. Furthermore, she urged us to think about which reaction we wanted to catalyse biologically and which reaction would be better done using chemistry.
These considerations had a direct effect on our part design which we made compatible between cyanobacteria and
Another challenge we had to address was how to get the modified cyanobacteria.
Cyanobacteria cultivation - Conjugation
Prof. Dr. Annegret Wilde is the leader of the research group at the Albert Ludwig University in Freiburg in the department of biology. Her research focuses are molecular biology and genetics of cyanobacteria as well as phototaxis and photoreceptors.
The most important thing when working with cyanobacteria is to avoid contamination. In principle the handling is not especially difficult but you have to be careful to use the correct light source and light intensity. Conjugation with triparental mating allows the creation of modified bacteria within a month and is much faster than using transformation techniques. Regarding the general project idea, Annegret Wilde doubted that coupling of ferredoxin to CYPs is really useful, since ferredoxin might not be able to maintain its activity in a fused state and additionally just the overexpression of the CYP might be enough to enable biosynthesis.
We implemented the useful input regarding the cyanobacteria conjugation to optimise our protocols. We were also a bit concerned that we might not be able to show an activity increase in the ferredoxin fusion proteins and therefore looked into possibilities to use knockdown strategies as control experiments.
Another important part of our project was the design of the linker sequence which connects CYP and reductase. We wanted to aid the design of these sequences by bioinformatic approaches but were unsure where to start.
Design linkers with bioinformatics
Prof. Dr. Andrew Torda is a research group leader for biomolecular modelling at the University of Hamburg at the Center of Bioinformatics. A previous team of iGEM Hamburg has worked with Prof. Torda and now both sides were excited to work on this project together.
We learned that for designing fusion proteins some parameters have to be considered. One of them is the selection of suitable linkers between the two different domains. Direct fusion of two domains could lead to misfolding and aggregation of domains which impairs their bioactivity. Suitable linkers can be designed using the knowledge of natural linkers. Using homology based sequence alignment a suitable linker sequence could have been found and tested in the laboratory.
By implementing these linker sequences the project profits directly from this outreach, increasing the likelihood of finding a suitable linker sequence.
Human Practices Beyond
Good Scientific Practice
While designing research projects you not only have to look into if your research project has a positive impact on the world but also obeys the standards of good scientific practice. If you do not keep that in mind all of the positive impact your project would have had on the world vanishes.
Since scientists are mostly measured by their contribution in adding new knowledge to science, there is a relevant danger of designing research in a biased way so that you receive the results you want. Therefore, guidelines are needed to ensure the quality of research. Young scientists should be encouraged to think about what these key aspects of good scientific practice are and they need to be able to identify and react to misbehaviour. In a workshop we learned about those standards and created mindmaps about these aspects of science. [verlinke 2 png Dateien in deutsch]
Keeping all of these aspects in mind we realised during our project planning phase, that some of our experiments, which should prove that our parts work, needed to be redesigned to ensure that their results are usable in a scientific context. We furthermore decided to separate experiment execution and analysis to avoid biased interpretation of the results. Without focussing on good scientific practices we would have not been aware about this issue.
Making a positive impact on the world and including all stakeholders
How are Artemisa annua farmers affected from our project?
All iGEM projects should focus part of their efforts in determining if their project is responsible and good for the world. Focussing on the antimalarial drug artemisinin it seems logical that decreasing prices by introducing a competitive micro-organism based production method has positive impacts and would be beneficial for a lot of people. But you also have to consider that at the moment artemisinin is produced by extraction from a plant which is mainly grown by farmers in low-income countries. What happens to those people if the microbial production is cheaper and companies no longer need this plant? Are there alternative income sources for those people, if they cannot grow Artemisia annua anymore? This concern was raised and we wanted to talk directly to the farmers to find out what they think about our project idea. Unfortunately we were not able to connect with those farmers, since we were not part of their local community. We discarded trying to reach the farmers through the company to which they sell their product, because we expected this company not to be very much invested in the interests of these people. Unfortunately, we could not resolve this issue which should not be forgotten if our project ever reaches industrial scale production.
Bill and Melinda Gates Foundation
Additionally we reached out to the Bill and Melinda Gates Foundation which has founded similar projects in the past. Our aim was to find out if they might be interested in funding our project. Additionally we wanted to find out their mindset toward bioeconomy becoming a major role player in the future. But unfortunately we did not get a response to our inquiries.
How can we optimize our project for large-scale production?
Another stakeholder we wanted to connect with was the industry. There are some interesting startups working with terpenoids and also global food, agrar and pharma companies are complementing their portfolio with projects in this direction. We were interested in finding out if our project could be implemented on a large industrial scale and how we could do something to make the transition easier. Also we were wondering if it would be possible to use cyanobacteria in this context. We hoped to get feedback and find potential collaboration partners for the future. Sadly, we did not hear back from several companies we contacted and could not organise a meeting with them.
Approaching the public
We also discussed approaching the broad public but discarded this idea because our project would have only a very abstract effect on their life. We could have conducted some surveys determining the general mindset of the public towards bioeconomy but considered this as not especially useful for integrating in our project.
Making our research accessible for everyone - inclusive web design
Finally, it is important to include everyone in the process of designing a responsible project. Disabilities should not lead to marginalisation and that is why we aimed to design the wiki website for our project as inclusive as possible. We seeked help from a company who professionally design inclusive websites and asked them for guidelines we could follow for our wiki. These guidelines should enable us to create an easy-to-use website with a clear and well-structured layout.
Creating a diverse iGEM team
Diversity is an important part of a functional iGEM team. When deciding which project ideas are good for the world, having diverse backgrounds, abilities and opinions is important. We are proud to be a diverse team and part of our work like the LabGallery would not have been possible without this diversity.
© iGEM Hamburg 2021