C O L L A B O R A T I O N S

Through this iGEM year we interacted with many iGEM Teams all around the world. It was a pleasure working with all the different teams and learn from each other. We got many impressions about other projects and we hope that we could help with our collaborations. We would like to thank every team that collaborated with us!

C O L O N Y
P I C T U R E S

In our colony picking collaboration we asked other iGEM teams to supply us with data for our picking algorithm. Thanks to this group effort we could build our Colony Picking Unit.

Pipetting Robot, colony picking set up — Figure 1: Pipetting Robot (OT-2) colony picking set up.

Many repetitive tasks in the lab cost time such as pipetting or picking colonies from agar plates and could technically be performed by robots. However, most commercially available solutions are simply too expensive and therefore unavailable for many Labs and iGEM Teams. So one of our goals was to build a reasonable colony picking robot to outsource the tiring task of colony picking by hand.

We used an Opentrons OT-2, the cheapest available pipetting robot and constructed modules and scripts, that allow other iGEM Teams to turn their own OT-2 into a colony picking robot as well. See here how to transfer your OT2 into a Colony Picker.

We chose to build an Artificial Intelligence (AI) to automatically recognise colonies. While a “hard-coded” solution would be easier to achieve, they lack accuracy and flexibility, key attributes of Synbio projects. The drawback for an AI approach is the amount of data it needs for precise and robust training. With the help of the iGEM-community we thought to overcome this problem. We asked other iGEM Teams if they could provide us with labeled pictures of their agar plates with E. coli colonies, which served as the basis for our AI. For that purpose we set up a Website.

To participate all that was needed were agar plates with E. coli colonies, a screen and a camera. Other criteria were evenly distributed, distinguishable colonies. The first step was taking the picture. For this purpose, the screen had to be set to the highest level of brightness and display a white image. When you take a picture of a common screen, a pattern on the picture could appear, the so called Moiré pattern. To avoid this, we supplied each team with diffusive sheets, which they could use to cover their screen. A detailed summary is found here.

Figure 2: Pattern for taking the colony picture.

colony picking website — Figure 3: Website for colony picture labeling.

Following this, the images taken had to be labeled using a web-based tool. Afterwards, the single colonies were marked with a rectangle. In total 250 colony pictures were added to our data set, vastly increasing the performance of our AI in detecting colonies. As an incentive to participate in this collaboration we gave away prizes to the teams that sent us their pictures.

Participants and Prizes

As an incentive to participate in this Collaboration we gave away prizes. The five best teams that sent us pictures got an E. coli plushie and the three teams that sent us the most pictures got a bigger surprise. Thanks to Doulix, we provided these three teams with an engraved trophy out of glass and vouchers.

Team BOKU-Vienna was able to send us one picture, team GO Paris-Saclay provided three pictures and team TUDelft could help us with 14 colony pictures. Team TU Darmstadt could send us a number of 23 pictures, but were beaten by team IISER Bhopal which send us an impressive amount of 99 pictures.

Prizes colony picture — Figure 1: Giving out the prizes for the best participants of the Colony-Picture Project at the giant Jamboree.

We thank all the teams for sending us labeled colony pictures. Without your help we would not have been able to train our AI and to transform our Opentrons OT-2 into a Colony Picker.

T H E G O L D E N G A T E

C O L L A B O R A T I O N

Our team made itself the goal to introduce other iGEM Teams to Golden Gate. Therefore we hosted a webinar, build up a communication platform and made an Interlab study.

Earlier this year the iGEM headquarters announced changes in their part submission system, one of which made us extremely happy: BioBricks are now also accepted in Type IIS standard! In our opinion, this is a huge step, as cloning techniques based on Type IIS restriction enzymes are becoming more and more popular in the scientific community due to their numerous advantages over classical cloning methods.
We noticed that more teams converted to MoClo systems in their projects this year but we still think there is a lot that needs to be done for the iGEM community to really embrace this change. In order to help teams with the transition into this new system, we had an idea: What if we could help other teams understand what Type IIS enzymes really are and how they work? If we could share our passion for Modular Cloning and the value of the PhytoBrick standard? If we could introduce them to our Marburg Collection and help removing some of the burdens that come with a switch to a new cloning system?
Therefore we hosted a webinar about Golden Gate, build up a communication platform to give iGEM Teams a chance to troubleshoot their methods and made an Interlab study. Here we evaluated the consistency of Golden Gate Assembly protocols across iGEM teams as well as to give teams the opportunity to gain experience with the assembly method.

Webinar - Educating about Golden Gate cloning

We wanted to reach as many teams as possible with our webinar on Golden Gate cloning, Type IIS enzymes, their benefits and how to use all of this in their own projects. We started planning everything that we thought should be in the webinar, from the biological function of the Type IIS enzymes and how they differ from Type II enzymes, over its applications in Synthetic Biology like modular cloning to the in-depth strategies when designing your own parts and actually building them in the lab.

During this planning step we realized, that some of the teams that are already working with Golden Gate might not benefit as much from an entry level webinar. Consequently, we thought about integrating more complex design questions that we could share with the community. We finished our webinars with a practical part, showing the part-design in Geneious Prime – the sequence analysis software we used throughout the year. Firstly, we explained the different levels that can be built with Golden Gate and then introduced some more complex designs that are also possible with Golden Gate cloning, such as the construction of whole operons or space holder parts. Through this we hoped to also reach teams that use Golden Gate themselves, showing them the possibilities this cloning system offers.

To reach as many teams on the globe as possible we set up webinars on three different times to cover as many time zones within a comfortable time frame – hoping that this will allow more iGEM teams to participate. The one and a half hour webinars were held on July 30th 10am CET, July 31st 6pm CET and the last time on 8th August 12pm CET by our instructor René Inckemann with the support of three of our team members. We were happy that in total 18 teams participated, as well as some students from the universities of Marburg and Gießen and even a few fellow scientists from the Max-Planck-Institute for terrestrial microbiology. In total we counted around 70 participants. We were also really happy to see Sota Hirano from Doulix on our webinar, who gave us some awesome feedback, stating that he did learn new things about Golden Gate which he was not aware of before!

Bilder Webinar — Figure 3: Participants watching our Golden Gate webinar all over the world.

If you would like to learn about Golden Gate assembly please feel free to watch our webinar through this Link:

We thank all the following teams for participating in this awesome event:

Slack Channel and gDrive - Global networking in the Golden Gate community

Although we were quite pleased with the result of our webinar, we thought about how to further connect this Golden Gate community, so we created a Slack workspace for everyone that is working with this method, enabling us all to exchange protocols and ideas or troubleshoot issues that might arise. So far there are close to 50 members, but we hope to find more enthusiasts willing to share their knowledge!

All files used for the webinar are openly available. You can find our protocols, handouts, slides as well as the files we used for the practical part in Geneious to construct parts in-silico in our shared Google Drive. Through this we hope to make it easier for other teams willing to start with Golden Gate cloning, as they can start designing their first own in-silico parts with our guidance.

The main documents of the Golgen Gate Webinar
→ Download: Golden Gate webinar slide 2019
→ Download: Handout Golden Gate webinar
→ Download: Golden Gate protocoll

To try out the described in-silico cloning you can download the needed sequences as geneious file here.
→ Download: Sequences Webinar

Interlab - Accelerate Golden Gate Assembly

As we are constantly working on improving the efficiency of Golden Gate. We clearly saw the main bottleneck of the reaction was the long assembly time in the cycler. In the webinar and in our Golden Gate community we provided protocols with three different cycler settings for different applications. Although these three protocols were tested before, they were never compared to each other in their efficiency.
We conducted our own Interlab study in collaboration with the iGEM Teams from TUDelft , HUmboldt Berlin and TU Kaiserslautern In order to find the best protocol, we supplied the teams with specific Level 0 parts,that should be assembled to a Level 1 vector using the three different cycler conditions. The provided parts are shown in the table

5' Connector	BBa_K2560075 /
Promoter	J23119 BBa_K2560031
RBS	B0034 BBa_K2560008
CDS	sfGFP BBa_K2560042
Terminator	B0015 BBa_K2560035
3' Connector	BBa_K2560080
ORI:	ColE1 BBa_K2560036
Antibiotic-Resistance-Cassette	Kan-RFP BBa_K2560056

The Golden Gate reactions should be pipetted together using the given parts and then be transformed in E. coli and be plated with similar amounts on agar plates. A colony with a correct assembled vector would have a functioning transcription unit which expresses GFP, which can be clearly distinguished from RFP expressing colonies which only carries the antibiotic cassette and false non fluorescent colonies.
The ratio of green to non-green colonies was used to evaluate which of the three protocols yielded in the highest transformation efficiency and the highest rate of positive clones.

The handout with the used protocols can be downloaded here .

Results

First we wanted to know which protocol worked best for every team. For this we analysed the data of the correct assembled green colonies for every protocol. The data of the different teams are given in Table 1 and are shown in as chart in Figure 2. The total colony count is hard to compare directly, cause the numbers differ very much between the teams. This could come through the different transformation efficiencies for example because of the variating competent cells the teams used.

Table 1 - Data for the gained green colonies gained by using the three different protocols, from each participating team.

Amount of gained green colonies gained by using the three different protocols, from each participating team. — Fig.2 Amount of gained green colonies gained by using the three different protocols, from each participating team

The results in Figure 2 show, that the protocols work differently for each team. In general the most colonies were received with the long Troubleshoot protocol, but also the Rapid and Improved protocols show high numbers of correct colonies.
But which protocol is the most efficient one? To compare the results of the teams the ratio of the gained correct colonies between the protocols were averaged. The data are shown in Figure 3 and show, that the long Troubleshoot assembly works the best. The fastest assembly (Rapid) works the second best. This is very interesting, because this assembly takes significant less time.

We would propose to take the Troubleshoot protocol for complicated assemblies and the rapid protocol for easy assemblies like lvl 0.

Fig.3 Average efficiency of the three protocols.

B E R L I N

D E L F T

U C D A V I S

UC Davis iGEM team is one of the winners of the Opentrons competition in 2019. iGEM Marburg won one OT-2 during last year’s edition of the competition and got contacted for a collaboration by iGEM UC Davis along with other winners from 2018 and 2019. The goal for this automation InterLab collaboration was to collect data to determine the accuracy and reproducibility of the OT-2 compared to humans. To do this, UC Davis proposed to perform the standard iGEM protocol on serial dilutions with silica beads and fluorescein, each manually and with an OT-2. In total iGEM Marburg did four dilutions; twice with an OT-2 and twice manually. The data was then sent to UC Davis via the spreadsheet designed by them. Finally, iGEM Marburg aided by creating a Slack channel for all iGEM teams with an OT-2 to facilitate better communication and exchange in the future.

UCD OT2 — Figure 1: OT-2 setup for the UC Davis collaboration

In total, seven teams participated and sent their data to UC Davis which are shown in the figure below. According to UC Davis, the fluorescence data was standardized using the iGEM MEFL conversion to standardize the plate reader measurements. Subsequently, the data was fitted using a linear regression model, whose quality is quantified using the r2 metric (it ranges from 0 to 1 with higher value indicates a better fit).

For the fluorescence the r2 value from the OT2 data beat human r2 value with 0.94 and 0.90, respectively. Interestingly, for the silica beads, manual measurements yielded a more satisfactory result with r2 value of 0.90 compared to 0.82 from OT2 measurements. One possible explanation could be the speed of the OT-2: it took twice as long to complete this task with the OT-2 compared to a manual trial. This could lead to the sedimentation of the silica beads as the OT-2 went through their runs; therefore worsening its results. Also, as shown in Fig. 2b, one team’s result deviates a lot from the rest of the teams, maybe if their result is taken out from the overall evaluation a more comparable result between OT-2 and human could be observed.

More information about this collaboration such as individual team data can be found on UC Davis wiki.

UCD Graphic 1 — Figure 2: a) OT-2 vs manual fluorescein measurement experiment, OT-2 performed better with r2 value of 0.94 compared to 0.90 of human. b) OT-2 vs manual silica beads measurement experiment, human performed better with r2 value of 0.90 compared to 0.82 of OT-2.

UC Davis Graphic 2 — Figure 2: a) OT-2 vs manual fluorescein measurement experiment, OT-2 performed better with r2 value of 0.94 compared to 0.90 of human. b) OT-2 vs manual silica beads measurement experiment, human performed better with r2 value of 0.90 compared to 0.82 of OT-2.

U F L O R I D A

I N T E R A C T I O N S

W I T H

O T H E R T E A M S

Mike the microbe

Mike the Mircrobe — Figure 1: Mike the mircrobe in our lab.

We participated in the “Mike the microbe” collaboration of the iGEM Team US AFRL CarrollHS. They sent us their virtual mascots Mike the Microbe and Chia the Chitinase so we could take pictures with them to connect all iGEM teams around the world via social media. It brought us great joy to join to this collaboration and let the two little buddies visit our daily life in our incubators for cyanobacteria.

Postcards Düsseldorf

iGEM Düsseldorf called for their traditional collaboration project, a long year postcard campaign with the purpose to educate and to show the public about the different topics and projects in Synthetic Biology and especially regarding all the iGEM projects. With interesting designs and a short project abstract iGEM teams could spread their ideas using the postcards to share their thinking to each other and the society. Our title and postcard design was about our photosynthetic project „Syntex“. You can see Synechococcus elongatus UTEX 2973 direly needing a refreshing drink, a cute little draft from our team member Jonas.

Postcard challenge — Figure 2: Our Postcard for the Postcard collaboration from Düsseldorf.

iGEMxSDGs challenge

SGD Challenge - 7 12 13 15 — Figure 3: SDG challenge.

IGEM Costa Rica, Tuebingen, and TAS Taipei created an iGEMxSDGs challenge as collaboration. They asked us to choose four of the Sustainable Development Goals (SDGs) proposed by the United Nations (UN). We joined this collaboration challenge and spread it on our social media account. Our project targets the goals shown in Figure 3.

Who wore it best? #labcoatchallenge

The Stony Brook iGEM Team challenged us to design an outfit for a runway from a standard lab coat. The outfit had to be created by tying, not using duct tape, cut or sew, etc. After designing our outfits four of our members presented their style on our ‘runway’ while paparazzi took their photos. As an outfit we chose a dress highlighting the neckline, a shoulder free dress, a knotted dress and a skirt to bring glamour to the daily routine in the labs. Especially our guys were rocking their outfits as you can see in Figure 4.

Figure 4: Our model Burgis for the labcoatchallenge.

M E E T U P S

iGEM Spring Festival in Bonn

From the 3rd to the 5th Mai we met other iGEMers at the Spring Festival in Bonn. Professors of the university of Bonn presented their research fields which was met with huge interest. We heard lectures about immunology and neurosciences, as well as reviving mammoths. During the lectures, specific topics were deepened in small seminar groups, such as the ethical tenability of CRISPR applied at humans and other animals. At the poster sessions we got the first time in contact with other teams’ projects. Afterwards, we had the opportunity to vote for the best project, which iGEM Düsseldorf won. Besides that, the meetup was filled with a lot of awarding categories. We competed in the categories for “Best Video”, “Best Poster”, “Best Project” and a single person had the chance to win the award for “Best Meme”. The iGEM Team Bonn offered spontaneously a little football table tournament, where two of our team members won. In the evening we learnt a lot about Bonn as a city from a guide living there for many years. Subsequently, the iGEM Teams met in several groups in restaurants and bars. We merged with iGEM Kaiserslautern and exchanged our projects and ideas for the rest of the evening.
On the second day, the iGEM Team Bonn offered a competition out of three rounds, where we competed against other teams. We were asked iGEM specific questions, had to solve riddles for an escape room and finished a lab challenge afterwards. Our team could compete until the end and won the prize! Thanks again to Doulix for this awesome prize!
We particularly thank the iGEM Team Bonn for the huge effort and the great meetup!

Erlangen meetup — Fig. 1: Two representatives of Doulix celebrating with us.

Erlangen collaboration meetup

On the 17th till the 19th of May we attended the collaboration meetup hosted by the iGEM team Erlangen 2019. There, we met the iGEM teams of Straßbourg, Tübingen, Vienna and of course Erlangen. Apart from listening to the interesting talks from the various professors there, we made a lot of new friends and took the chance to talk about possible collaborations with the other teams and also promote our collaborations, such as the colony picking project and the Golden Gate webinar. We would like to thank iGEM team Erlangen very much for organizing this wonderful meetup!

Düsseldorf meetup

From the 5th till the 7th July we went to the HHU for the German meetup. Many lectures with interesting topics were offered by the Cluster of Exellence of Plant Science (CEPLAS) by Prof. Andreas Weber but also non-scientific aspects like courses on presentations. We tried to visit all of them while presenting our poster in the hall. There we got in touch with other iGEM Teams and also learnt about their projects. We promoted our Webinar project and laid the foundation stone for future collaborations. On the second day, iGEM Düsseldorf organized a panel discussion about the issues concerning green genetic engineering. Thereby, we got great input on how to structure our panel discussion and which subjects should be mentioned. Beside the regular program organized by iGEM Düsseldorf we used a lot of chances to get in contact with other teams. After the presentations a couple of tourist guides showed us the beautiful old town of Düsseldorf and explained some interesting historical facts. Afterwards we had again the possibility to meet with other iGEMers in a pub and talk about the projects, especially to find weaknesses or opportunities for collaborations. This really went successfully due to the time we spend with iGEM Team Aachen, Kaiserslautern and Hamburg. The iGEM Team Düsseldorf also arranged a game contest between the teams, where we lost in the final to our good friends from Potsdam. Congrats!
Thanks again to iGEM Düsseldorf for the huge effort and the great meetup!

Du meetup — Fig. 3: Team photo from the Düsseldorf meetup.

Vilnius, Lithuania – Biohackathon Lab App 2019

Vilnius biohackathon — Fig. 4: Biohackathon in Vilnius.

In August 2019 our Team participated in the Biohackathon Lab App, organized by the Vilnius-Lithuania Team 2019. Together with the iGEM Teams Stockholm and UCopenhagen, as well as several non-iGEM Teams we sat together for a weekend packed with problems, ideas and neat solutions. With the task of creating an application that tackles a self-found problem, we sat together and brainstormed about what could be done.
During this time we were starting to work more with the Opentrons OT-2 and soon realized that some of our biologists had trouble integrating protocols in the robot themselves, as this is primarily done by writing the protocol in Python and pushing it directly to the robot – no convenient application to change parameters, such as the amount of reactions,was included in the protocols. In our heads the idea to have a user-friendly GUI grew more and more and soon we found ourselves standing in the Vilnius University Life Sciences Center.
“We” were two biologists and one mathematician, so we talked to some of the other participants that did not have a team yet and found the perfect fit to complement our small team: Nour Alsamsam was happy to join us for this weekend and provided us with his coding skills – huge thank you to you, Nour! Together we tried our best to come up with a suitable app and presented it in the end, having more ideas in our heads than we could implement in such a short time – it was clear for us, that we would continue with this project later on!
In conclusion, the Biohackathon was an awesome and productive meeting of the iGEM teams Stockholm, Copenhagen, Vilnius and Marburg. We talked about our projects, offered each other feedback on current issues in the lab and shared many ideas. We are very grateful that we got the opportunity to meet so many experts from different fields, which really helped getting a fresh perspective on our project!

Team:Marburg/Collaborations

C O L L A B O R A T I O N S

C O L O N Y P I C T U R E S

The Colony Picture Collaboration