mCherry Collaboration with CeBiTec Bielefeld

For scientific networking, we have contacted the iGEM team CeBiTec from Bielefeld. They prepared experiments with mCherry, which is a fluorescent protein and and TexasRed, a red rhodamine fluorescence dye, and since we also work with fluorescence proteins such as mVenus or sfGFP, we were able to agree on a way to combine this knowledge and gain new experiences in alternative fluorescence proteins. They established a protocol to extend the repertoire of standardized fluorescent proteins from GFP, which is already extensively standardized via fluorescence, to a second reporter protein, mCherry. For the different experiments, the plasmids and an aliquot of TexasRed were kindly provided by iGEM Bielefeld. The obtained plasmids contain a gene coding for a chloramphenicol resistance as well as several strong constitutive promoters. The plasmid maps are shown in Fig. 1, indicating that the gene for mCherry were cloned in the iGEM vector pSB1C3. Three different promoters from the registry were used to control expression of mCherry: BBa_J23104, BBa_J23114 and BBa_M13108.

**Fig. 1**: Plasmid cards from the constructs J23104 (A) + mCherry (red arrow), J23114 (B) + mCherry (red arrow) and P8Prom.(C) + mCherry (red arrow). The plasmids contain an chloramphenicol resistance cassette and all containing promoters are constitutive.

**Fig. 2**: Agar plates with pSB1C3 + mCherry in Escherichia coli BL21. The colonies show a red color.

Escherichia coli DH5α and BL21 (DE3) were transformed with the different obtained plasmids and plate on LB-Agar containing chloramphenicol. The red-fluorescent colonies on plate were observed by eye and were helpful for a fast detection of positive clones. Positive colonies were inoculated in LB-medium containing chloramphenicol. The cultures were grown for 16 h and the absorption was measured and the cultures diluted to an OD₆₀₀ of 0.02. The diluted cultures grew for 6 h before 200 µL aliquots were pipetted on a 96 well plate for the measurement. Three biological and three technical replicates were made. A serial dilution of TexasRed was added to the 96 well plate. The serial dilution started with 1 mM and a 1:10 dilution was made ten times and can be seen in the Fig. 3.

**Fig. 3**: Serial dilution of the red rhodamine fluorescent dye TexasRed. The fluorescence was measured at an excitation wavelength from 570 nm and an emission wavelength of 620 nm. The linear equation shows a slope of 18340 ± 97.27 [AU/mM] and a high maximum fluorescence.

**Fig. 4**: Responses of fluorescence were compared between E. coli DH5ɑ and BL21 (DE3). The strengths of the different promoters J23104 (red and blue), J23114 (light green and light blue) and P8Prom (yellow and dark green) were tested. The fluorescence was measured at an excitation wavelength from 570 nm and an emission wavelength of 620 nm.

The Fig. 3 and 4 show that the the fluorescence response of mCherry is weaker than the fluorescence response from the serial dilution of TexasRed. The highest fluorescence from the mCherry experiment can be compared to a concentration between 1 and 2 mM of TexasRed. A absolute response from nearly 1800 could be measured. Fig. 4 shows that E. coli DH5ɑ has a higher production of mCherry than E. coli BL21 (DE3). The measurement also showed that the promoter J23104 has a stronger production of the reporter gene mCherry than P8Prom, whereas P8Prom has a higher production compared to J23114.

Beside the protocol from the iGEM team Bielefeld, another experiment was made with the constructs. The real-time fluorescence of growing bacterial cultures was measured over 20 h using a plate reader (Tecan). This experiment was performed to quantify the fluorescence development over time at different growth phases, including the lag and log phases.

**Fig. 5**: Responses from fluorescence over time from E. coli DH5ɑ (A) and BL21 (B) transformed with the different constructs containing the aforementioned promoters. The fluorescence was measured at an excitation wavelength from 570 nm and an emission wavelength of 620 nm. The J23104 shows a higher gene expression rate than P8Prom and higher than J23114 in general.

Fig. 5 shows a similar result compared to the endpoint measurement from mCherry and the serial dilution. The fluorescence response from DH5ɑ is higher than from BL21 (DE3). The obtained data also indicates that the promoter J23104 is the strongest promoter in the applied setup among the investigated three promoters and the P8Prom. is stronger than the promoter J23114. The progression of the curve of the BL21 J23104 is surprising. At the beginning of this curve, the fluorescence increases a lot, but after nearly 2 h the fluorescence starts to decrease and remains on this reduced level.

Collaboration with UC iGEM Team New Zealand

During the research for our project we came in contact with UC iGEM Team New Zealand. The team aims to develop novel strategies to increase the value of milk proteins that will, in the future, be produced using cellular agriculture. To be more specific, five protein variants of the milk protein β-Lactoglobulin will be engineered to be more digestible, hence less allergenic for human consumption. Since we are in the same track and share the interest of synthetically creating milk components, what better way to share ideas and help each other by collaborating! We instantly got in contact with the team via Instagram and scheduled a skype date, despite the eleven hours time difference. Our first collaborative skype meeting was on July 3rd, where we discussed the outlines of our project, social outreach methods and more. Being from a university that participated in the competition three years in a row, we were happy to help the first time iGEMers and shared our knowledge that we gained in the past. We had great talks and directly agreed on not just one, but two ways to collaborate!

We conducted a 3-month survey of our project to understand the valuable point of view of as many people as possible. Therefore, we promoted our survey on Instagram. We asked general and specific questions in the questionnaire. The main focus of the survey was to find out the general viewpoint of people on climate change, the environmental impact of different products, the estimated impact of livestock, the importance of eco-friendliness of products, if the participants payed attention to non-GMO labels when buying groceries and how often they consume cow's milk. The results were then shared with the UC iGEM Team to have a better global sense of how the community feels about the projects.

Furthermore, one of our subteams focused on the production of the whey proteins α-lactalbumin and β-lactoglobulin. Since the New Zealand team concentrated on β-lactoglobulin we shared the idea to exchange them. This means: We sent the variant α-lactalbumin in pET22b(+) vector while we received their β-lactoglobulin variant in pET-Duet. Both teams aspiration was characterization and/or gene expression with each other’s gene. While the UC iGEM Team analyzed our construct, we expressed their construct in Escherichia coli BL21 (DE3). The results were successful on both ends, which on our end is shown by successful expression of β-lactoglobulin (~25 kDa, Fig. 1).

**Fig. 1**: Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Escherichia coli BL21 (DE3) secreted protein. The SDS-PAGE was run at 200V for 40 minutes and then stained with Coomassie blue overnight. β-lactoglobulin has a molecular weight of 22.4 kDa. 1) not induced sample, 2) induced sample cell lysate 3) induced supernatant 4) induced wash supernatant 5) Empty 6) not induced EVC 7) induced EVC cell lysate 8) induced EVC supernatant 9) induced EVC wash supernatant.

UC Davis Opentrons Collaboration

We participated in the interlab opentrons collaboration by the iGEM Team from UC Davis (UCD). With the robot our team won in the last year and the protocol provided by UC Davis, based on the 2019 iGEM Fluorescein Calibration Protocols, we were able to execute a pipetting challenge: Human vs. Robot!

For this challenge, a team member, and the opentrons robot (OT-2), conducted a serial dilution of fluorescein in a 96-well plate. Subsequently, the data was measured using the BMG Labtech CLARIOstar plate reader. The raw data achieved by the team member is shown in Fig. 1 and the robot’s data in Fig. 2.

**Fig. 1**: Raw data of the serial dilution of fluorescein prepared by a lab member. The measurement was executed in the BMG Labtech CLARIOstar plate reader at 485 nm (Excitation) and 535 nm (Emission).

**Fig. 2**: Raw data of the serial dilution of fluorescein prepared by the OT-2 robot. The measurement was executed in the BMG Labtech CLARIOstar plate reader at 485 nm (Excitation) and 535 nm (Emission).

This dataset was sent to the UCD iGEM Team, which evaluated it to a combined standard curve of all the participating teams (Fig. 3; left) and a standard curve for each team (Fig. 3; right).

**Fig. 3**: Left: Combined fluorescein standard curve (log.) of all teams and all robots combined. Right: Fluorescein standard curves for each team and each robot separate. Both graphs are based on the raw data the teams sent to UCD. iGEM Düsseldorf = Team 7.

The data indicates that, compared to a human, the robot is more accurate when pipetting the serial dilution (Fig. 3), since the data points of the same dilution lay closer together when prepared with the robot than done by a human. This represents a strong mechanical limitation, which is based on human nature. The challenge implies that robots may be more accurate in pipetting, butcan they do memes?

International Postcard Project

As we all know, iGEM is not only about our projects, but also attaches great importance to engaging the public with synthetic biology. In order to do so, the previous iGEM teams of the Heinrich-Heine-University Düsseldorf created a project to promote synthetic biology in public by using self-designed postcards. Each year, the amount of participating iGEM teams has grown, with a big stack of beautiful and funny postcards from all over the world as a result. We wanted to continue this international community effort once more and asked the iGEM community to be a part of this collaboration, which turned out to be a huge success.

**Fig.1**: Distribution of postcards to various teams worldwide.

How does the postcard exchange work?
Every participating team had to design a postcard which shows an image related to synthetic biology or their project on the front and a small informational text on the back. The postcards were sent to us, mixed and then sent back to each team.

We reached a lot of iGEM teams by showing off postcards from the previous years on Instagram and other social media accounts and were also the first ones to post on the collaboration page of iGEM 2019. In addition to that, we also collaborated with iGEM Stockholm, who presented previously designed postcards at their art exhibition in an old nuclear reactor in Stockholm, Sweden. Another external collaboration partner included @igem_memes, an instagram account that posts funny memes all around iGEM and therefore unites all iGEMers in their struggles, despair but also fun times. They gifted an iGEM meme postcard to every team, which was highly anticipated by all iGEM teams. Additionally, After iGEM also joined our project.

A lot of iGEM teams responded who wanted to channel their inner creativity and design their own postcard. We had 60 applications and 48 participating teams in total, making it our biggest collaboration (and the biggest iGEM collaboration in general?) yet. The first postcards arrived during the iGEM MeetUp from iGEM Potsdam. From then on, we eagerly waited for the mail to arrive every day and were often rewarded with packages containing new, beautiful postcards from all over the world. One package travelled around entire Europe before finally finding its way to us in Düsseldorf.

After they had all arrived, we had to take a day off to sort through the postcards, pack the parcels and bring 48 packages to the post office. Once all postcards had made their way into the whole wide world, all teams were able to share them with people in the neighborhood, during public events etc.

All Postcards:

Art Exhibition – Collaboration with Bielefeld

“Was die Welt im Innersten zusammenhält” (translated: “Whatever holds the world together in its inmost folds”) was the theme for the art exhibition we organized with the iGEM Team CeBiTec Bielefeld at the Goethe-Museum Düsseldorf after they got in touch with us and suggested a collaboration like this.

**Fig. 1**: Picture of the art exhibition. **Photographer: Hans-Peter Skala.**

A few weeks before the event took place, we had a meeting with Damian Mallepree, the curator of the art exhibition to prepare and discuss our project. We talked about the Faust-Labor. It is an exhibition about Goethe’s Faust in all its facets. Johann Wolfgang von Goethe was a famous German author who lived from 1749 to 1832. The book “Faust” is one of his well-known creations. It is about a scientist named Faust, who searches for the meaning of life.

For the art exhibition we created exhibits to the history of synthetic biology and the modern laboratory and merged Goethe's concept of the homunculus with our project ideas in the iGEM competition. The homunculus is an imitation of a human out of organic material. Goethe was convinced of the existence of a “lifeblood” which is in all creatures to divide organic and mineral material. The theme of the homunculus is combined with the idea of a successful natural sciences.

With this possibility, we wanted to link synthetic biology with lyricism, by merging both subjects together, to engage people with an interest in poetry and art rather than science. Synthetic biology have a long history which can also be seen in features of Goethe´s stories. His interests in natural sciences influenced his writing. The idea of a synthetic life is today present in the search of the minimal organism as part of the synthetic biology.

We started to plan this collaboration early to reach a successful art exhibition. On the 18th of May, we have met with the curator of the museum Damian Mallepree and members of the iGEM Team CeBiTec Bielefeld. Damian welcomed us with drinks and snacks and together with iGEM Bielefeld CeBiTec, we started to brainstorm what exactly we want to show to the visitors. The basic framework was formed and the project took a final shape. It was good to see what exactly Damian, as someone who does not have a connection to biology or the everyday lab work, imagined for the art exhibition in contrast to us, as future scientists with no good connection to lyricism. In fact, this kind of interaction was our goal - on the one hand, to reach a lot of people, with no connection to biology as it first seems; on the other hand, to learn about non-scientific issues from other people, which can be affected by genetic engineering.

**Fig. 2**: Successful meeting with CeBiTec Bielefeld at the Goethe museum.

On the 17th of July, we officially started our art exhibition. At the beginning of the opening ceremony, talks were given about Goethe and Synthetic Biology. The first talk from Dr. Heike Spies and Damian Mallepree was about the character Faust and his work as a scientist as well as Goethe's interest in the natural sciences which influenced his stories. After this, Prof. Dr. Jörn Kalinowski from the CeBiTec Bielefeld held a speech to the topic “Goethe's 'Faust' and the Synthetic Biology". He showed the basics behind synthetic biology and the sequencing of DNA and made a connection to the story of Goethe's 'Faust'. At the end of the opening ceremony the iGEM Team from Bielefeld and we presented our projects in short talks and received many questions and positive feedback.

**Fig.3**: Mirko, a member of our team, presenting our synthetic milk project at the opening day of the art exhibition.

After the talks, we initiated the real art exhibition with live-experiments. With snacks and drinks provided by the museum, we welcomed the guests to the exhibit which included self made posters about the beginning on the research of DNA, everyday laboratory work methods and, of course, about topics related to the image of the homunculus by Wagner of Goethe’s Faust or the primary plant mentioned by Faust. Beside of the experiments we had the opportunity to have personal conversations with people, who are not familiar to the work in the laboratory and the research behind it.

This exhibition was an amazing possibility to connect to people outside the laboratory and create a collaboration which can be developed in the future for future teams.

“iGEM Squeeze” - The iGEM board game

While we were working on finding our 2019 iGEM project, we had a team building evening on the 29^th of March, 2019 and played the board game “iGEM Squeeze” from the iGEM team of Rotterdam. This board game is made to help other teams find more ideas for their projects. The goal of the game is to select between good ideas that are possible and ideas that are not interesting. The manual on how to play the game can be found on iGEM Rotterdam´s wiki.

**Fig.1**: Some of our members playing the iGEM Squeeze board game to find ideas of our new project.

Important for the decision are the questions “why?”, how?” and “what?”. With these three questions in mind, it was easier for us to think about the way we can present our project. On the other hand, we were able to find questions about our topic that we did not have in mind but could have been asked by other people.

At the end of the first selection, we had three possible projects to choose from. The topics were synthetic milk, cannabidiol (CBD) production and soil improvement. After a long discussion, we decided that we would not do soil improvement because the game helped us realize that this topic could be too difficult to sell. We also did not continue with cannabidiol after we felt certain that we wanted to have something real to show. In the case of CBD, we were sure that we would be able to market it very well, but the implementation of the science would not be simple at all.

With the synthetic milk project, we found a topic which has many parts to work on and therefore is more related to practice to better show our results. Also, the section “Nutrition” is important for us as a German team, because we further took it to our heart to change the German people's view on future food and the potential use of genetically modified organisms. We hope that we may contribute to reducing the negative connotations associated with products derived from these organisms.

In the end, the board game “iGEM Squeeze” also helped us to find our own ideas to introduce new people to iGEM to find better into the competition. We believe that the mindset behind the board game is very interesting and will also help future teams to find very good and interesting projects. Furthermore, this game helps new teams to get closer and start communication more quickly among individual members.

Meetup

**Fig.1**: Group picture of all participants.

For a comprehensive report on our German iGEM MeetUp, check out the extra page.

Synthetic Biology Art Exhibition

The communication of science especially in the field of synthetic biology is a big part of our job as young, upcoming researchers. For this task, many new and innovative approaches can be made, so iGEM Stockholm decided on a creative approach by organising an art exhibition.

This way, synthetic biology can be shared with everyone because art knows no limits. Since we love sharing the benefits and beauty of science with the whole world, we wanted to contribute to this event. During the last three years, the past iGEM teams from Düsseldorf organised a postcard exchange project where all interested iGEM teams designed a postcard related to their project, team or synthetic biology in general to create a visual impression for people who are not familiar with science. These postcards were then exchanged between the participating teams and were also handed out by every team themselves.
Over these years many amazing designs were created that we wanted to share with a broader audience. We sent the generated postcards of the previous years to iGEM Stockholm so they could display them during their art exhibition. Everyone was amazed by the great postcard designs and the variety of projects that the iGEM competition had authored all these years.

Skype Meeting with iGEM IISc-Bangalore

Day after day we work with simple and established model organisms like Escherichia coli or Saccharomyces cerevisiae. We wanted to choose the best fitting organism for our purpose of protein production. With the help of the research center Jülich we came to the conclusion to use a special Bacillus subtilis strain which has already been used in big scale protein production. Since the work with this organism differs in some points from the handling of E. coli, we made an effort to get more familiar with it. Since we were well informed, it was a pleasure for us to teach iGEM IISc-Bangalore all the little tricks and important facts they needed to know about B. subtilis during a skype interview. They also planned to integrate B. subtilis into their project and also asked for some tips which strain is best for their case. We were also able to connect the team with our last year’s iGEM Düsseldorf 2018 team so they could exchange knowledge about co-culturing and cultivation in general. We were so pleased to help iGEM IISc-Bangalore out so they could get started immediately without figuring out which way is the best to reach their goals.

Team:Duesseldorf/Collaborations