Expertise from the research center Jülich
Since we are students of the University of Düsseldorf, we are very fortunate to have access to the research centre in Jülich, which takes on a leading role in new biotechnological techniques and methods. They are a tremendous source of expertise in all matters of the biotechnology sector.
Production of desirable proteins is a large factor in biotechnology as well as in our project, so it was a great opportunity to talk to experts which are working on protein production. At the beginning our organisms of choice were Escherichia coli, Pichia pastoris and Synechocystis. During our exchange Dr. Andreas Knapp recommended Bacillus subtilis as a production organism, since it will be able to secrete our protein of interest directly into the growth medium. This would simplify the purification of the proteins since we will have less problems in getting rid of bacterial residues, which is crucial for a food product.
The conversation also dealt with the right use of different protein secretion tags which are special DNA sequences, coding for certain amino acid sequences (Signal peptides), which that lead to the secretion of a protein when they are fused to it.
In addition to their advice and experiences, they provided us with the B. subtilis DB 430 strain, which is very well suited for protein secretion, as it is missing several proteases, that are secreted in the wild-type2. They also provided us with the pBSMul1 plasmid including five different secretion tags targeting the proteins we fuse to them for secretion, so that we could try out which signal is working best for our milk proteins.
Dr. Knapp also gave us the advice that there is no secretion sequence that works for every protein, so trial and error is the only option to find optimal sequences. We took this advice to heart and designed several plasmids for a big experimental setup that aims to find the best suitable secretion tag for every single one of our proteins.
We were also provide with feedback on our project, our ideas for production, the general handling of B. subtilis and Dr. Knapp gave us insights into experimental design when scaling up our production.
The input of this meeting with Dr. Knapp from the research center Jülich convinced us to switch the main production of our proteins to B. subtilis to ensure the possibility of large scale production in addition to the other two organisms. E. coli and P. pastoris will still be used for, the first, protein analyses and Synechocystis has the advantages of CO2 neutral production which enables eco-friendly production, which would be optimal considering the main motivation of our project is to reduce greenhouse gases.
Meeting with a leading company in synthetic dairy production
We had a telefon Conference with the CEOs of a leading company in synthetic dairy production. They even currently released their first product, an animal free ice cream.
Climate change is the most discussed topic in the whole wide world these days. It is a topic everyone is engaged in and no one can run away from. One of the big producers of greenhouse gases is the dairy industry. The amount of methane the cows extrude are enormous. Even though we produce way less methane by farming than CO2 it has an 28 times higher impact on the climate, causing a huge impact on the greenhouse effect1.
The CEOs of the American startup company, saw this problem years ago and were determined to start a change. Their idea was simple: Producing dairy proteins by microorganisms for vegan milk.
By now they have build a big company and have assembled a large team of researchers that is really close to deliver a finished end product. They recently announced that their completely cow-free and cruelty-free milk will be available in just a few years and released their own vegan ice-cream made of microbial proteins, which was sold out in just a day.
They company is planning on expanding the production to other dairy products everybody craves, such as cheese or yoghurt. And everything without harming millions of animals or producing massive amounts of dangerous greenhouse gases. Isn't that amazing?!
During the research for our synthetic milk we discovered this company and since we share the same goals and basic ideas, we reached out to the company and were very lucky to be able to talk directly to the CEOs.
We were grateful for their huge expertise in the field, as well as their kindness and sympathy towards the problems that occurred during our work. They gave us many good advices. For example which organisms we cloud use and how we could handle them. Additionally they gave us useful input, on which other animal products could be produced by microorganism in the future, like egg proteins, so that we have new ideas what we could do in the next iGEM years. We discussed large scale production and how much time and research the finished product needs with them as well.
Since we both are aiming to create the same product we wanted to make sure that we do not get in law conflict with them, since they patented some of their production methods of proteins in microorganism, but the patents did not state the exact methods. Thankfully, they gave us the permission to use whatever organism that they patented to produce our proteins to optimise the systems and supported us in trying out and experience the benefits and disadvantages of the organisms we choose. For the production of fatty acids we had no thematic overlaps with them, since we want to produce our fatty acids in microorganism as well and they do it in plants.
Further into the meeting we spoke about possible ingredients of milk to improve the taste and consistency for it to become as similar as cow’s milk. During their research the start up came across many different ideas of what could be added but because milk has many different components that need to be optimised for large scale production, and they were not able to focus on the development on every upcoming idea. They shared the idea with us to produce the milk protein lactoferrin, which they do not focus on in the moment, but are interested in. With this they gave us the opportunity to broaden the spectrum of our targeted proteins and to collaborate with them in research. After the meeting we decided to produce lactoferrin, since it is known to have antibacterial and anti carcinogen effects, which would be a beneficial ingredient for our synthetic milk, and started researching right away.
With the help of this company team and constant consultations we will be able to implement the production of lactoferrin in Pichia pastoris into our project to improve our milk of the future. We are very excited for the opportunity to be able to collaborate with such an awesome company and be part of the change we both want to see in this world!
Daniel Ducat
Daniel Ducat is a well known cyanobacteria expert at the MSU (Michigan state university), who was already able to give us valuable input and new ideas for the iGEM competition in 2018. This year we didn’t want to miss the chance to talk with him about our project and the usage of cyanobacteria for the generation of synthetic milk.
He provided information regarding the carbon footprint of cyanobacteria, in order for us to adjust our project towards an environmentally friendlier production of the product. Additionally, he introduced us to different application areas of the single components to be found in our SynMylk. For example, short chain fatty acids can not only be used in our synthetic milk, but in the cosmetics industry as well as for the production of biofuels. This indicates that the individual components of our SynMylk already have a great benefit not only for nutritional purposes.
After the meeting we decided, that we wanted to try to produce different components of our SynMylk in cyanobacteria, namely short chain fatty acids (C4 to C18) and proteins, to have an organsim creating the products, that can maintain its life cycle and production of the components by photosynthesis to decrease the carbon footprint of our product. Without Danny´s input we might not have decided on using cyanobacteria but on other expression systems for our production. His expertise about the system and the hint that they can already produce a broad range of lipids supported us in the decision making.
Another important topic of our conversation was outreach work. As there are strict regularia in Germany regarding the use of genetically modified organisms for food production, he gave us great ideas on how to go public with a project such as synthetic milk.
The main idea was to find out when customers would stop buying products like food. Whether this happens with the mere mention of the word ”bacteria” or whether genetic engineering must be explicitly mentioned. This project was not carried out due to time problems, but is still pending for the future.
Talk with the StartUp Expert Eugen Kaprov
After studying the synthetic food market and analysing our survey, we quickly realized the great potential of SynMylk for becoming a real product after iGEM. Therefore, we were happy to find a partner in “SYSTEMIC VENTURES” (SV) - a venture capitalist corporation focussing on synthetic biology.
On May 27th, our team met with the representatives of SV, presenting our project, giving a market outlook on the milk industry and showing the possible scalability and impact behind our idea.
Subsequently, they introduced us to the financial background of startups: financing funds, tools and their orders of magnitude, possible life cycle scenarios and the risks of the different starting phases, but also to other relevant issues like the customer-feedback-loop. The risks were pretty humbling, but helped us aquire a more rational view on our plans.
We were interested in receiving more knowledge about the foundation of an enterprise and the background in this area. We discussed many questions we had and they showed us how a startup can strengthen its position inside the economy. In addition, they gave us a summary of risks and characteristics inside the biological sector.
Together we discussed the possibility to transform our project into a realistic startup and received many ideas on how we could manage it. We came to the conclusion that a startup would be a great possibility for our project in the future and that it would definitely be doable with the right partners.
Promega Webinar
Thanks to the cooperation of Promega and the iGEM headquarter, we were given the chance of a high-quality webinar series, where our team learned and asked about experimental struggles. The Promega scientists explain biological topics and give advice on how to deal with possible problems. Furthermore the webinar implieds a question and answer session, staffed by technical services scientists and webinar speaker. Webinars are an important tool for everyone in iGEM. Since it is an international competition it is a great chance for everyone around the world to participate and learn from experts from different countries. Long journeys involve travelling time and expense, but a webinar eliminates the need to travel.
Within the 3 webinars thematizing ligation, transformation and transfection we got plenty of professional and efficient advice, for example about the best way to prepare the vector and insert for ligation, the need of including a control when transforming or variables to regard when transfecting mammalian cells. For our team the tips were very useful, since we had problems with the individual cloning steps, to be more precise the transformation of Bacillus subtilis for our milk proteins, we received great advice (Fig. 1). The webinar was a perfect fit for our time schedule, as at that moment we were working on the transformation.
Again, we wanted to thank both iGEM HQ and Promega, for this awesome possibility.
Doulix
We want to thank Doulix that they gave us the chance to improve their user-experience! We used their product and gave valuable feedback and this further showed us how important User-Experience feedback is for improving a product. Therefore we want to establish it as a key evaluation tool in Entrepreneurship.
