HUMAN PRACTICES
Silver criteria
Visit from Forensic Biologist Dr. Mark Benecke
Dr. Mark Benecke, a known forensic biologist not only in Germany but also worldwide, did not only work for the FBI, but also became a forensic expert for biological traces after moving back to Germany. Therefore he plays an important role in solving problems related to crime in our country.
Dr. Benecke immediately caught our attention when he gave a talk at the March for Science in Cologne, as he is an open-minded and interesting person. His research field, his profession and especially the way he teaches people about science encouraged us to invite him to our university to present our work and our lab.
Luckily, he accepted our invitation and visited us on August 27. We had a very extensive and captivating conversation with him, receiving great advice regarding our story, our project and our way of presenting ourselves. By encouraging, us he motivated us to make it to Boston and made us even more enthusiastic about iGEM. We had a lot of fun, especially when recording a video about synthetic biology and our project for the public. This way we created a funny, informative and an internationally accessible brief description of our “Trinity”.
Since Dr. Benecke is an influencer known worldwide, we hoped that synthetic biology will receive a broader public reach and engagement due to his visit to our laboratory.
Our successful meeting with Mark Benecke can be seen on the social media platform of our university and is also visible on Mark Benecke's facebook page, ensuring that synthetic biology, the idea behind iGEM and our “Trinity”, is spread.
We would like to thank Dr. Benecke for his support and his thoughtful words. We really appreciate his input and learned a lot from his advice.
We hope that we were able to reach a wider audience with our collaboration with Mark Benecke and were able to show that synthetic biology is not dangerous, but on the contrary, very fun and helpful.
A Survey for Everyone
To assess the public’s opinion regarding a future possibility to utilize our toolbox for the synthesis of probiotics, we created a survey with a couple of questions.
We did not only share this survey on our social media accounts for a short time, but also went to one of Germany’s biggest supermarkets to personally ask customers what they think about genetically engineered probiotics. In total we achieved a participation count of 267 including young community members starting from 14 years and up to older people. Among the participants, male and female people were almost distributed equally.
Most of the participants were relatively young, ranging from 14 to 30 years old, which is why we considered that the vast majority of the survey was filled out online. Because of the results from question 3, seen in Figure 1, we assume that most participants are students, probably biologists and familiar with synthetic biology and its hazards. In contrast to that, most of the people we addressed at the supermarket were older, between 30 and 50 years old and frequently even older (+60 years old).
This observation is further enhanced when looking at the answer of question 3: Here most of the participants admitted to having an academic degree. Most survey attendants at the supermarket did not hold an academic degree, but mostly rather had a lower secondary school level or no degree at all.
Interestingly, independent of age, gender and degree, most of the participants are aware that the intestinal flora has an impact on human health. This conveys that everyone has already heard something about the gut microbiome and the fact that there are indeed living organisms somehow working to improve our health.
However, when looking at the most frequent answer to question five, some of the participants have never consciously taken probiotics to improve their health. When asking this question in the supermarket, most of the people did not immediately know what probiotics are or what they consist of, until we named some known examples of probiotics and explained them that they harbour microorganisms. This outcome is very peculiar, as most of them are aware that our gut microbiome plays a huge role in our daily life, yet they do not consciously try to improve their health by for example consuming probiotics.
We believe that this result is due to the lack of trivial information in everyday life which we realized when we encountered a woman suffering from Crohn’s disease in the supermarket. Even though she belonged to the older subgroup of participants, she was very well informed about synthetic biology, GMOs and potential harms. Her age however did not keep her from approving of the consumption of genetically modified organisms if this would improve her health.
Moreover, also when considering the answers of question 6 it can be clearly seen that for us seemingly simple questions like: “What do you consider a genetically modified organism?”, were frequently answered incorrectly. This became obvious to us at the supermarket, when looking at the people struggling to answer this question.
All in all we observed a trend at the supermarket during our chats with the customers. We got the impression that young adults with higher academic degrees tended to be more aware of GMOs. However, similar to the other participants, they rejected the consumption of GMOs as well, even if this would improve their health.
This clearly indicates that the public is still not very familiar with synthetic biology and its potential benefits.
The last question was whether people would agree to the consumption of genetically engineered probiotics. With our survey and with our personal interaction with the public at the supermarket we can conclude that there is a tendency and acceptance of people to consume synthetically engineered probiotics. We had the impression, that the ones denying the consumption of genetically modified organisms had a distorted image of synthetic biology due to lack of public knowledge regarding this issue.
Cyano 2018: 3rd Early Career Research Symposium on Cyanobacteria
One of the major key players in our co-culture is Synechococcus elongatus, a cyanobacterium that provides the other organisms with carbon.
Every year a conference on cyanobacteria takes place in Germany where experts meet and discuss recent progress, research and their current projects. Last year it was held at our university in Duesseldorf, which is why our advisors knew of it and advised us to visit the conference this year.
We sent the organizers of this symposium an abstract of our project and were actually accepted to present our project in a 20 minute talk.
On the 12th of September two of our members started their trip to Freiburg, a beautiful city in the South of Germany, where the 3rd symposium for cyanobacteria was held for three days. We were greeted with a lot of sun and temperatures up to 80 °F (30 °C), whereas the other members of our team had to freeze at 15 °C in our lab.
Every day a couple of talks were held in addition to a presentation from a keynote speaker.
On the first day a team of Serbian scientists presented their research on cyanobacteria in their natural habitat. Slađana Popović even found a new species in a cave in Serbia! Christoph Howe then talked about his idea to use cyanobacterium biofilms for H2 production for biofuels.
Another interesting idea was the one of Moritz Koch. He presented his idea of cyanobacteria as a PHB producer - a carbon friendly bioplastic against the pollution of the sea on the second day.
In between poster session and discussions we were able to socialize with other cyanobacteria researchers. The organizers even offered a guided tour to the Münster - a famous church in Freiburg and a hike to the Schlossberg (castle rock) which offered a great view over Freiburg and the surrounding hills.
The last day of the conference was the big day for us: We got to present our co-culture project in front of the real experts of cyanobacteria.
After explaining what the iGEM competition is to a lot of the people who hadn’t heard of it, we started detailing our concept around the cyanobacteria in the planned co-culture.
After the talk the audience ended up starting a discussion about our strategy. We are very thankful for the mentioned advice and tried to integrate all the input in our work.
Expert Discussion with a Pharmacy Employee
The gut flora is a very important network of organisms in our bodies. Itie has a big impact on our overall health, wellbeing and recovery. Just like other microbial consortia, these networks have not yet been extensively studied. Even though it is such a significant aspect of our health affecting drug absorption we try to establish synthetic co-cultures with our toolbox that can be used in many diverse fields. We thought it would be a great idea to talk to an expert about it. Hence, we invited Mrs. Küpper, an employee of the Paracelsus pharmacy.
After quickly introducing us and iGEM we gave her a presentation about our project and our goals with it for society.
Just like others Mrs. Küpper was very interested and asked many questions about our co-culture.
Especially impressive to her was the fact that the co-culture toolbox can be used in many possible ways - either for production, the cultivation of different organisms, simple research or as probiotics.
She informed us that probiotics currently arouse not only the interest of companies but also of her customers. Every week new products emerge on the market and are sent to the drug store. A lot more people also come to the pharmacy and ask for probiotics against diarrhea or stomach trouble even without being sent by their physicians. A lot of doctors still prescribe antibiotics even for minor illnesses which could be treated otherwise, thus accepting the damage of their patients’ gut flora. Some customers use probiotics to counter the side effects. Some physicians - unfortunately not all - recommend probiotics to their patients.
Mrs. Küpper also noted that most of her customers are aware that probiotics are living organisms which is why they often ask for the correct storage of the medicine. Dead organisms are used as a sort of vaccine to start the immune system - but then they are not called probiotics anymore.
Since they are not subject to medical prescription the customers need to pay for them themselves. German health insurance doesn’t cover the costs.
In the end she also suggested other applications of our toolbox. The differently built, but similar functioning networks of organisms also exist on the skin or in the mouth that - being impaired - can also cause illnesses. According to her our toolbox could provide important research for those as well.
Synthetic Biology and Waffles
Our idea behind the small information booth paired with waffle sales was to capture the general knowledge of the students of our university about synthetic biology while “baiting” them with delicious self-made food.
We organized a quiz with general questions about microorganisms, genetic engineering and molecular biology where everybody was able to participate.
It was interesting to see that biology seems to be very complicated and incomprehensible to many. Many students who were not familiar with biology at all, often failed at the quiz but then were cheered up with a delicious waffle and every information they asked for. Some terms like amino acids and DNA were not familiar to a bunch of participants, but few were able to put the terms into the right context. However, there were some students who had solid knowledge in synthetic biology - despite studying something else - and were also interested in the iGEM competition itself. We were happy to share our knowledge with them and to draw them under the spell of biology while handing out more waffles.
A few other students were already familiar with the iGEM competition itself and visited our booth to find out what this year's topic is about. We were delighted to present our project and were also able to inspire some non-biologists with our idea.
All in all, we were able to arouse interest in synthetic biology with our information booth and were able to enjoy a bunch of waffles.
Hopefully we were able to contribute to bringing the wonderful world of synthetic biology to many new people and to take away their fear of this often unknown science.
Radio Interview
The [97.1] Hochschulradio Düsseldorf is a universitarian radio station hosted voluntarily by students of different study fields. Even though there is a lot going on at the Faculty of Mathematics and Natural Sciences the station only has seldomly the chance to welcome guests with a scientific background which is why they were very happy to invite us. Our team members Salima and Carina were interviewed by Cigdem Ünlü on June 18 at their radio station.
Our goal was to change their problem, introduce iGEM to a bunch of folks who listen to the station and advertise the upcoming talks of our lecture series.
When we entered the station we were quite surprised: It’s located in a normal apartment in a big residential block for students. After we were greeted we began right away with asking a lot of questions since we were pretty nervous: Are we prepared enough, what language style should we use, how should we deal with bloopers and who was going to say the important stuff. But it turned out that they were actually quite easy going so we had nothing to worry about.
Cigdem began asking us many questions as soon as we met to prepare for our conversation. Once she was ready, our interview began and everything was broadcasted live.
At first we were asked about the iGEM competition to give the audience an idea about what it actually is for. iGEM might be well known among scientific faculty members and science students at our university but students in other fields of study are usually not familiar with the term.
After the quick introduction to the competition we started explaining synthetic biology in more detail - which is a quite unknown expression as well - and gave examples of what we’re actually dealing with next to our research, such as sponsoring, meetings with experts, modeling and spreading the world about synthetic biology.
This was a perfect transition to our past and upcoming outreach projects that Salima quickly presented. We invited all fellow students to come. Our interviewer was quite happy to know that those events were also for non-scientist without any knowledge in synthetic biology.
Next we had to answer why we joined the iGEM team and mentioned practical experience, our own project and exponential growth eeh learning curves as our motivation.
This led to our current project - the co-culture consisting of three organisms. We tried to explain our plan in a way that was also understandable for people who didn’t know much about biology. Cigdem asked whenever something was too complicated which gave us a great sense as to what might be hard to understand when you’re not dealing with it everyday and every night.
Before the interview ended we were once again allowed to advertise our upcoming lecture series talks on the radio.
All in all it was a very interesting experience to talk in front of people you can’t see which gives you a rather odd feeling. We learned how to describe a complex project to people with little knowledge about specific mechanisms and were able to reach people beyond our usually scientifically interested audience.
V-Bio Karl-von-Frisch Award Ceremony
Every year the VBIO - an association to promote biology - awards excellent high school graduates in Biology a prize. Just like the previous year the iGEM team Duesseldorf helped to welcome the students and gave a talk about the iGEM competition in general and their project.
Four members of our team took the train on June 26 and arrived at noon at the BioMedizinZentrum (BMZ) in Dortmund. After meeting Professor Susanne Bickel and the organiser, we helped them distribute badges to the students in order to mark them as guests. Some cakes and beverages later, Dipl.-Ing. Michael Kuhn started to give a short introduction about the center. Professor Susanne Bickel then explained the VBIO association, with their goals and tasks and made some advertisement for it, showing the advantages for students.
After the students came back from a tour that led through the center, it was our turn to present.
We explained the iGEM competition in detail, presented efforts and project ideas of other teams, showed what our current work in the laboratory looks like and presented goals we have in mind for our project. Afterwards, we were drilled with questions. The students showed a lot of interest in the iGEM competition and were curious about possible teams to join. Even a few teachers chimed in.
In the end we had an interesting discussions with some teachers and students regarding our project ideas and our progress.
We left feeling happy about the motivation and inspiration we were able to give.
Visit of Fabian Nienhaus - Karl-von-Frisch Laureate
At the Karl-von-Frisch award ceremony we got to know a high school graduate who was awarded for his excellent achievement in biology. He wrote an outstanding term paper, in which he co-cultivated Bacillus subtilis and the yeast Yarrowia lipolytica. One of the hypotheses he offered for the behavior he observed is that the co-cultivation forced one population to produce an antibiotic substance as a defense mechanism.
At the Karl-von-Frisch award ceremony he expressed interest in the iGEM competition and our project. Because he still had numerous questions regarding our co-culturing project after the event, we invited him to visit our university and our lab. That is why Fabian traveled the long way from the Münsterland to Düsseldorf to get more insight into our project.
In order to give him a tour of our working places and laboratories, we gave him a complete safety instruction, as required by the lab we work in. Armed with a lab coat and safety goggles we explained to him, among others things, general techniques used in laboratory work with nucleic acids and microorganisms and showed him our beautiful cyanobacteria.
Since our project and his term paper cover closely related topics we had a great discussion about similarities like the use of co-cultivation, but also differences, for example symbiotic vs. combating combinations for co-cultivating.
The time flew by fast and soon Fabian had to start his way home. He will always be welcomed in our lab and we wish him success in his pursuit of a medical degree.
INTEGRATED HUMAN PRACTICES
Gold criteria
Cyano and Co-culture expert Daniel Ducat
In the beginning of our work with the three “Trinity” organisms we stumbled across several questions:
How should the growth media be composed?
How can we effectively use our cyanobacterium Synechococcus elongatus as a sugar producer?
How can we prevent the other organisms from overgrowing S. elongatus?
The synthesis of sugars in S. elongatus, especially aiming at a co-culture with heterotrophic organisms, has previously been shown by Dr. Daniel C. Ducat from Michigan State University. His Synechococcus cscB strain produces and exports sucrose in salt stress media. During our brainstorming sessions at the beginning of our iGEM journey, this strain was often named as a main example for the use of cyanobacteria in a co-culture. Finally, we decided to contact Dr. Ducat and asked him for advice and good ideas regarding co-cultures with cyanobacteria.
Luckily, he was on a symposium visit in Germany in May 2018 and offered to visit us and provide us with the cscB strain. In a private meeting, he suggested to use a sps (sucrose phosphate synthase) overexpressing Synecchococcus strain. Thus, we would be able to have sucrose production and export, without having to induce high salt stress in the culture media with NaCl. Although sucrose production would be less effective we could prevent the negative effects of salt stress regarding our heterotrophic bacteria and fungi.
In one of Dr. Ducat’s publications (A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production, 2017), he used barium alginate beads to encapsulate cyanobacteria to enhance sucrose export. We were interested in using these beads as another option for co-culturing methods besides solid and liquid culture. Dr. Ducat provided us with a protocol for creating these beads and suggested to encapsulate all of our “trinity” organisms.
Most importantly, he suggested M2 as a co-culture medium to us, which is derived from the cyanobacterium medium BG11. He also provided us with the protocols which were then frequently used in our project .
In summary, Dr. Ducat helped us significantly in several ways that changed our project. At first, by providing us with the Synechococcus cscB strain which enabled us to conduct experiments with sucrose production and export in salt stress media.
Second, by introducing a co-culture medium to us which we ended up using for all of our co-culturing experiments.
We are glad to be given the opportunity to speak with a co-culture specialist and value his advice that changed our project significantly.
Integrated Human Practices with Dr. Spencer Scott
Our work with the quorum sensing system is based on research done by Dr. Spencer Scott and colleagues, who published the paper “A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis” in the well known “Nature microbiology” journal in 2017.
Since we knew we would encounter some difficulties and had questions regarding this harsh project, the best choice for us was to reach out to Dr. Scott in order to improve our project.
At first we presented him our current progress and he showed himself very delighted that his PhD project was further studied by us. He also assured us his support.
Our idea was to use the same microfluidics system that the researchers used in the paper mentioned above. Since the paper does not precisely state which kind of system they used, we asked if their system was easily reproducible in our lab. Unfortunately, Dr. Scott informed us, that their lab was endowed with very sophisticated and costly microfluidics system and advised us against this setup. Instead he suggested us to use a simple chemostat.
Moreover, he suggested to try to apply the quorum sensing systems in different organisms, since the used parts should, according to him, be transferable to other organisms.
In addition to that, we encountered some issues when comparing the growth situation of the wild type co-culture with the mutant co-culture which included the quorum sensing harbouring microorganisms. He recommended to just check the overgrowth of the specific organisms in the two different situations.
Regarding this, another important advice he gave us was that a good overall experimental time is 24 hours.
Finally, to solve the problem of quantifying AHL - the quorum sensing molecule of interest - he proposed to us to use a qualitative approach instead of a quantitative one, hence to try titration of different AHL concentrations and to check the impact it has on our cultures.
When getting to know Dr. Scott, we found out he was a member of the iGEM team Berkeley 2011 which is why he did not only give us significant advice for our project, but also told us about his experience with iGEM, further increasing our enthusiasm for iGEM and the final Giant Jamboree.
We would like to thank Dr. Scott for this amazing skype interview and the great input he gave our team. We hope - with the gained iGEM experience and future research we will do a great job and one day will also be able to help other iGEM teams and play a role in their interesting projects.
Interview with Shraddha Shitut
Shraddha Shitut (Sh) is currently working as a postdoc at the Leiden Institute of Biology and institute of Chemistry in the Netherlands. In her PhD she worked on cross-feeding in bacteria resulting in a publication "Metabolic coupling in bacteria" in bioRxiv 114462 with which we found many similarities to our subproject "Auxotrophy". There she describes the establishment of a co-culture working with unidirectional cross-feeding with amino acids like lysine, histidine and tryptophan. We asked her for advice and wanted her to give an opinion on our approach.
TD: Hello Shraddha! We are iGEM team Duesseldorf 2018 (TD) and want to know, if you would consider giving us an interview and advice for one of our subprojects. For this year’s project we are working on establishing two- and three-way co-cultures between Escherichia coli and Saccharomyces cerevisiae and the cyanobacteria Synechococcus elongatus. One of our approaches is establishing a cross feeding of amino acids between a leucine-auxotroph E. coli and a lysin-auxotroph S. cerevisiae; they are producing the needed amino acid for each other. What is your first impression about our plan?
Sh: It is an ambitious plan. If we only consider the 2-way interaction between the modified E. coli and BY4742 you need to remember the additional auxotrophy in BY4742 and provide Histidine in the medium. This may affect the dependency of the auxotrophic E. coli strain. In my experience in some situations the auxotrophy can be fulfilled even by the presence of other amino acids in the medium. Hence it would be ideal to test of all auxotrophs that are to be used in the project in the presence of the different amino acids to be used in the project. There should be a clear difference here - growth when focal amino acid is provided, no growth when any other amino acid is provided.
TD: Exactly. Our Auxotrophy system is used in a two way co-culture. We have already added histidine but didn’t consider that it could influence the dependencies. So, Thank you for this tip! Why did you start researching auxotrophies and co-cultures?
Sh: I got interested in studying the interactions between bacteria (in a social or community context) during my studies for my masters degree in microbiology. Then I further read about metabolic dependencies in microbial communities and how this possibly affects the percent of bacteria we can cultivate and isolate. It was fascinating to learn how auxotrophies can lead to interactions and networks between microorganisms.
TD: Interesting! Where can this focus on co-cultures built on auxotrophies lead to in research?
Sh: Auxotrophies can be thought of as the dependency of an organism on an external source of an essential molecule which in most cases are amino acids, vitamins or nucleotides. These metabolites have to be part of the primary metabolism in a cell and hence essential for growth and proper cell function. A co-culture or interaction based on the exchange of such essential metabolites makes the interaction itself obligatory for the involved partners and hence a good model system to study. Furthermore due to the involvement of the metabolites, especially amino acids, in primary metabolism one can observe direct effects of the interaction (taking place in co-culture) on the behavior of both partners.
TD: Do you have experiences with amino acid exchange without exporters?
Sh: The cross-feeding system that I used and studied for my PhD did not inherently contain mutations in any of the amino acid exporters. We observed exchange of amino acids via physical structures connecting cells. However we cannot completely rule out the involvement of exporters as a response to amino acid starvation.
TD: In your publication it is mentioned that the exchange took place via nanotubes. Did you modify your cells for this? And if yes, how?
Sh: We used a synthetically engineered cross-feeding system wherein we introduced two mutations in wildtype
TD: We are working with a lysine auxotrophic system. Can you explain why Lysine, Histidine and Tryptophan are good candidates to work with ? Why are they so often in focus of studies?
Sh: Lysine, Histidine and Tryptophan are usually selected for their isolated biosynthetic pathways which allows for easy manipulation without affecting the production of other amino acids.
TD: What were the challenges you faced and what do you think could be challenges with our approach?
Sh: The first challenge of your project is to introduce the auxotrophies and amino acid production/secretion mutations in the different partners. The second would be to ensure that the produced amino acids are sufficient to support growth (Keep in that due to the fact that the partners belong to different classes their amino acid requirement levels will likely be different). The third challenge is to successfully cultivate the 3 organisms such that each partner remains stable over a time period long enough for you to study the interaction. Here you need to take into consideration differences in growth rates, nutritional requirements, ambient pH and so forth for the 3 organisms.
TD: What methods did you use to quantify your amino acid production?
Sh: I used both an indirect biosensor based approach and a chemical analysis technique (LC-MS) for quantifying amino acid production.
TD: What method would you suggest if we want to extract amino acids from the cells?
Sh: Internal amino acid extraction requires two main steps - breaking open the cell and purifying the amino acids within. The cell wall and membrane can be disrupted either by physical techniques (sonication, heat, beads) or by chemical techniques (lysozyme). The choice of techniques is dependent on factors like equipment availability in the lab, number of samples to be purified at a given time, characteristics of the cell (i.e. cell wall thickness) and so on. The second step of purifying the amino acids from the cell extract also differs based on which amino acids are of interest (free amino acids, protein bound amino acids).
The method I used in my study was based on a physical disruption of E. coli cells using heat and sonication followed by solvent based extraction of internal free amino acids. This method is useful for extraction of internal amino acids from numerous and multiple samples.
TD: Did you also measure the composition of your cultivation media to know how much of the produced amino acids are exported? If you did, can you suggest a method to use here as well?
Sh: I was using minimal media for all my experiments and during the chemical analysis of my samples for amino acid quantification I always used asterile control media. This was especially important because minimal media contains ammonium chloride as a nitrogen source and the NH-group here interferes with LC-MS measurements. More specifically because we were derivatizing (using FMOC to basically label the amino groups to make it detectable in the machine) the samples prior to amino acid quantification and we had to add excess reagent to first nullify the NH-groups already present in blank minimal media.
TD: We are working on a co-culture with different organism classes. Did you use organisms other than E. coli and what are your experiences?
Sh: We used Acinetobacter baylyi in the cross-feeder cocultures with E. coli. A. baylyi is a gram negative soil dwelling bacteria hence not very different from E. coli in terms of amino acid biosynthetic pathways and lab cultivation. It was however tricky to introduce the auxotrophic and over-production mutations in A. baylyi as compared to E. coli. This (genetic manipulation) I think is the biggest hurdle in organisms that are less established than E. Coli, followed by finding the right media that supports the growth of all partners.
TD: What do you think are the advantages of co-cultures and co-cultivation in general?
Sh: I think co-culture and co-cultivation approaches can give us a lot more information about bacterial behaviour than isolation based techniques. By culturing bacteria with other organisms we introduce a factor in its environment that can result in a number of reactions or interactions, both positive and negative. Hence such techniques can lead to novel compounds being produced by the involved bacteria.
TD: Thank you very much, for your precious advice and time. You helped us a lot!