Team:IISER-Tirupati India/Partnership
We met and collaborated with iGEM teams from all over the world and had numerous insightful, knowledgeable yet crazy moments with all our collaborating teams.However, we had a different connection with 3 teams and we collaborated with them beyond just the scope of one event. Our partnerships with 3 iGEM teams crossed all borders and oceans.
Our partners during iGEM 2021 were :
- Team Groningen from the University of Groningen, Netherlands.
- Team IOANNINA from the University of Ioannina, Greece.
- Team MIT_MAHE from the Manipal Institute of Technology, Manipal, India.
This page talks about how we had long term collaboration with all our partners and how this journey had an impact on our project.
Starting from our discussions on how to manipulate Saccharomyces cerevisiae for our collaboration to discussing how wiki freeze is driving us crazy. It’s been an amazing journey with Team Groningen and we loved to be a part of this roller coaster ride with them.
The page shows the landmark events in our partnership with Team Groningen during iGEM 2021 and Fig 1 is a representation of the timeline of our partnership which shows how extensively teams worked together and collaborated on different aspects of iGEM.
Now, here’s a detailed timeline of our monthly achievements and what we could achieve in the end, together with Team Groningen.
June
On June 9th 2021, we received an email from Team Groningen’s team leader, Ivelina Goldstein after they saw our message on the iGEM 2021 Global Slack. We were happy to find a collaborator working on Saccharomyces cerevisiae, and we met with them hoping that we may have some points in common. This way, we both could learn from each other without too much overlapping.
We communicated with them, and luckily, we met with Team Groningen for the first time on 16th June (Fig 3). In this meeting, both teams shared their project ideas.
Team Groningen presented us with their project, which we found to be an exciting idea. Their project aims to find a solution to the Dutch nitrogen crisis by preventing the release of ammonia by the digestive tract of cattle. They plan to genetically engineer S. cerevisiae to produce alpha-amylase and incorporate this in cattle feed.
We were excited to know about their project and see how we had some points in common even if our projects were so different in terms of topic track. Several points were covered, we discussed the following:
- Wet lab: We found out that they would also use S. cerevisiae and the Golden Gate Assembly standards for their project. We also came to know that they were going to use Bacillus subtilis (our chassis) to compare it with their chassis, S. cerevisiae.
- Dry lab: Their plans were to produce their protein in a regulated and well-monitored environment, while our modelling is based on a dynamically changing environment. Therefore, most of the modelling techniques we planned on using were not applicable to their project and vice versa.
Possible collaboration could’ve been achieved in the field of molecular dynamics and protein modelling. Our goals were to study the interaction between Ovastacin and ZP2 and, we would have collaborated on this front if we had started working on urease inhibitors for their project.
- Collaboration ideas: We were discussing the medal criteria Improvement of Existing Parts and we proposed that there are existing libraries for B.subtilis and S.cerevisiae parts in the iGEM registry that are not well-characterised, so we could figure out a way to collaborate on a part that we can improve and divide the work.
Since we didn’t have any lab access, we were trying to explore the possibility of a hybrid collaboration.
We discussed that we were using the same cloning technique (Golden gate), so collaborating on improving a part shouldn’t be too difficult.
This collaboration and most of the wet lab ideas were discussed with Ivelina, their other Team leader.
Conclusion:
- We were really happy to explore this possibility of collaborating on a Gold medal criteria Improvement of Existing Parts (wet lab collaboration)!
- We also looked into whether they could outsource some experiments to our lab in case they don’t have prolonged access to theirs (as we were certain that we would have a lab). Since they were certain that they would get lab access, we were grateful to hear that they could probably help us with some experiments since there was uncertainty with respect to the COVID-19 situation in India
- A dry lab collaboration would only be possible in the field of molecular dynamics.
Overall, the meeting was smooth, and both teams were very enthusiastic about helping each other. We had nice feedback from them.
July
We met again on the 20th of July (Figure 4) to discuss further the wet lab part of our projects. In this meeting, the members of the wet lab team from both teams joined!
We discussed our idea of using S. cerevisiae and our yeast construct. We discussed the chassis that both teams were using and had in common, S. cerevisiae and B. subtilis. This was such a relief and exciting turning point for us. We had found a team with such similarities even though the projects were so different.
We talked about the strains that both teams were planning to use, and we told them how heterogeneous protein production can be optimised in B.subtilis 168. We found out that the S.cerevisiae S288C strain is a suitable strain for cloning and protein expression experiments, and they were kind enough to offer us the strains for our experiments.
Milou from Team Groningen also brought up the topic of turning this collaboration into a Partnership, and we were quite delighted to hear this. Based on our discussions on our collaboration ideas and looking at the lovely and enthusiastic folks from Team Groningen, our team knew that they would surely be one of our partners.
Conclusions:
Several conclusions were withdrawn from this meeting.
- The most feasible option for a long term collaboration/partnership that we saw then was to compile data and characterise the promoters and terminators in S. cerevisiae that both teams will be using.
- We decided to share our protocols in Slack (Figure 5), so that one team can use the protocol from the other(as we were more experienced with B.subtilis and they were with S.cerevisiae).
- We learned some valuable information to use in our project. We found out that the S.cerevisiae S288C strain is a suitable strain for cloning and protein expression experiments.
- Both teams expressed their interest in a Partnership for the iGEM 2021 season.
After this meeting, we created a Slack workspace where we could share protocols, articles and doubts. This way all the members of the teams could take part in the collaboration.
August
Our monthly meeting happened on the 18th of August. In this meeting, we made the decision to extend this partnership beyond the wet lab and explore other avenues of working together. We, therefore, decided that we could work together on the following topics:
- Wet lab: We found out that our projects were differing quite a bit from one to the other, but we wanted to still work together on troubleshooting, as it had been beneficial in the past. Thus, we decided to meet once we start working with B. subtilis and we start with transformation experiments with S. cerevisiae, we could set up a meeting to discuss the challenges/tips the other team has learned so far.
- Human Practices: We invited them for recording an episode of our podcast “SynTrack”. Also, since we had conducted several surveys and had got them reviewed by our Institute’s ethics committee, we agreed to send them our surveys and the inputs that we got from the ethics committee. Team Groningen worked hard on an information sheet and informed consent sheet and sent it to us, which helped us a lot.
- Dry lab: Team Groningen told us their plans to use the ART tool and the AlphaFold2 pipeline to have a better insight into their device. They offered us help with these tools.
- Wiki: Since we have a master coder, Ankush, on our team, we offered them some help to overcome some minor bugs in their code.
During this month, we kept in touch through the Slack channels.
September
After having shared some nice conversations through Slack we decided to meet again on the 20th of September to check on each other. In this meeting, different things were discussed.
- We discussed about the content of the podcast we were going to record together. We decided to talk about synthetic biology in the food industry. After a good discussion of the topic and the format, we finally set a date to meet for the podcast!
- We then went through the typical wet lab discussion of our meetings. We talked about different ways of cell lysate preparation and different uses of the Golden Gate Assembly. Since we were running into some problems with our experiments, we tried to troubleshoot it with Carian and Sofia from Team Groningen. After this discussion, our experiments started working.
During September and October, we also collaborated by adding music to our team's shared open playlist which we listen during coding the wiki or post-lab sessions. Milou suggested many amazing additions to our playlist, and we were grooving on their beats throughout the month. The playlist can be found under the name iGEM wiki mix on Spotify.
October
- Finally, in October, Shubhra from Team IISER Tirupati sat down with Milou and Sofia to record our podcast episode (see Figure 6). In the episode, we talked about synbio in the food industry, their potentials, and the risks. We enjoyed the previous discussion we had with them about it, and we learned a lot about the topic. Team Groningen also gave a marvellous introduction to their iGEM 2021 project during the podcast.
If you want to know more about the episode, listen to SynTrack on Spotify!
- In the end, Shreyas met with Team Groningen (Figure 7) a couple of weeks before the wiki freeze. It was a nice meeting in which we talked about our experiences in iGEM so far, and we shared the feelings that involve having the deadline so close. In October, everything seemed to be around the wiki, and we weren’t aiming to be out of that; we discussed how to prepare the wiki pages and share coding experiences. We talked about the documentation of our partnership on our wiki’s and Team Groningen did an amazing job in making the timeline (Figure 1) of our partnership.
We are grateful to Team Groningen for always tolerating our silly mistakes and pushing us to do better and we’re thankful to their entire team for their useful insights in different aspects of the competition, from the wet lab through coding the wiki, to human practices. We are really glad to have shared this exhausting but unforgettable iGEM season with the team Groningen. (see Fig 7) Cheers to our amazing journey, cheers to our teamwork, cheers to us. We hope to stay in touch with them beyond iGEM and hope that our paths converge once again someday.
Last but not least,
All the best and we wish you all the luck and success!
Our first interaction
In the month of April , our co-team leader, Shreyas initiated the conversation with Team MIT_MAHE by contacting their team leader, Anooshka. They had typical conversations about team registrations, sponsorship and project ideas, of course.Then we initiated a conversation about planning the All India iGEM Meet 2021 with them.
One fine day, we saw their message on Slack and it was a really unexpected turn in this journey with MIT_MAHE. We came to know that they are working on Bacillus subtilis as well, and we immediately set up a meeting to discuss our projects.
On 17th of May 2021, we had our first meeting to discuss our project ideas and explore the possibility of using our model organism, Bacillus subtilis. After a long discussion, we decided that unless we get lab access and work on B. subtilis (which was common for both parties) there is not much room for collaboration
AIIM brings us back together
Our collaboration didn’t stop at just one meeting. We collaborated on organising the All India iGEM Meet (AIIM) which was held in the month of July. All the iGEM teams from India came together and hosted a bunch of events and presented their project ideas. Our teams bonded a lot during this process because of all the back-end work that goes into organising such massive events.
Wetlab team back on campus
Miraculously, our team got permission to get back to campus and get lab access in the month of July. We started our basic experiments in August and luckily during this time Team MIT_MAHE also got lab access.
However, being in an engineering college they had to face a lot of issues and they put up a message on Slack to enquire if any Indian iGEM team has the strains and lab access.
The rest is history. We contacted them immediately and helped us get in touch with Dr. Sabari to get the Bacillus subtilis168 strain and E.coli clones for the vector pDR111.We quickly signed a Memorandum of Understanding with them to mark our partnership.
Troubleshooting our way through the experiments
In the month of September, we tried our best to help them with troubleshooting some basic molecular biology experiments as we had worked with Bacillus subtilis for a few weeks. It was a fun and learning experience for us as we discussed lab work with them. We even shared our growth curve of Bacillus subtilis 168 with them data for us to compare. This was the time when the wetlab members from both the teams interacted the most and spent time discussing our work with each other. Megha from Team MIT_MAHE did a fantastic job in communicating with Shreyas and working out the details of this partnership on a weekly basis.
Wiki Freeze and Software Testing
In the month of October, our teams met to document our partnership and discuss how wiki freeze has got us all nervous. Laughter and happiness took all the anxiety away and we looked back to how both teams became partners.
Team MIT_MAHE has built software to produce 3D CAD models of bioreactors, BioREActorDesigner (BREAD) and luckily we were able to test it out for them and give our feedback. It is a very promising software for future iGEM team to look into and utilise for their project. (We loved the name “BREAD” )
How we summed up everything
In the end, all we have to say is that we’re grateful to have Team MIT_MAHE as our partners as it was a learning process for both teams and we had our moments of fun and joy! Working towards helping each other in every possible way was the motto throughout and it’ll continue to be the same.
We wish them all the luck and success!
Overview - How it all started
On 22nd June 2021, we saw their message for collaborating over biosafety aspects of the project. Our team saw this as an opportunity for a long term collaboration because biosafety is one the key values of our project. We contacted their team over Instagram and set up a virtual meeting to exchange ideas and discuss our potential collaborations.
In our first meeting, we discussed the general aspects of our projects. We planned future meetings to discuss the biosafety aspects of our projects, and we shared our project descriptions via email. We also formed a Whatsapp group where members from both teams communicated, bonded.
Kill switch - Our Wet Lab Design
Their project this year, antiBYEotic focuses on degrading Tetracyclines and Macrolides released into the environment, by genetically modified Escherichia coli. They needed a kill switch design for their project and we already had designed a kill switch for the biosafety of our GMO. So, after the first meeting, we agreed to share our kill switch designs and related literature with them.
Both teams decided to work collaboratively on the toxin-antitoxin, (bpDNase1 - mf-Lon) that our team had designed to work under blue-light induction. Team IOANNINA decided to incorporate our design of toxin-antitoxin system into their project design under the regulation of antibiotics present in the environment and agreed to test the shared system for us as we had limited lab access.
Even though the teams had different goals for this kill switch and we were working on Bacillus subtilis and Team IOANNINA was working with Escherichia coli, the design of this system made it modular and flexible to work in different organisms.
Since Team IISER-Tirupati_India had already worked out a design and the modelling of this kill switch, we shared the module with Team IOANNINA, it was decided that Team IOANNINA would help us test the toxin-antitoxin system in E.coli. This would provide proof of concept for our novel system and help team IOANNINA to build an efficient kill switch.
Here on, Kyrania from Team IOANNINA and Shreyas from our team had hours and hours of discussion about the design of experiments, modifications required to fit the need of both the teams without affecting its functionality, and thinking about how this can help both the teams in the best possible way.
The Design
The discussion between Kyrania and Shreyas are summarized below:
- Testing the system in E.coli and B.subtilis
- Initially, we were concerned whether testing our system in E.coli would suffice for proving the idea of this toxin-antitoxin system.
- Upon discussing this with amongst both teams, we realized that since the function of bpDNase1 and mf-Lon might be considered host independent. Their expression and activity have been shown in different chassis organisms independently, we concluded that if promoters of similar strength are used in different chassis, we predict that the system would work in effectively similar in E.coli and B.subtilis.
- Conclusion: Team IOANNINA will test the system in E.coli for the preliminary proof of concept.
- Checking the expression of mf-Lon and bpDNase1
- We were brainstorming ways to detect the proteins of interest and quantify their expression. We went through the possibility of using IMAC since Team IISER-Tirupati_India had some experience with this technique or HPLC or using reporter proteins like fluorescence proteins.
- However, we concluded that Team IOANNINA wouldn’t be able to perform these experiments due to limited access to certain lab equipment.
- Checking the functionality and interaction of mf-Lon and bpDNase1
- Team IISER-Tirupati_India suggested that we could check the functionality of this design by performing an experiment with the following setup
- Making a clone consisting of: expression of bpDNase1 under an inducible promoter and mf-Lon under a medium/weak strength Anderson promoter which team IOANNINA was already using.
- While performing the experiment, a control would be an aliquot without induction and the expression of bpDNase1 will be induced in different aliquots at different concentrations of the inducer and at different timepoints.
- Then the OD at 600nm or CFU of the cultures can be measured to account for the bacterial cell apoptosis due to DNA degradation caused by bpDNase1.
- Conclusion: Team IOANNINA would try the above experiments to test the system and execute them with required optimizations and Team IISER-Tirupati_India would be involved in the process of further experimental design and troubleshooting.
- Deciding the strength of promoters and ordering the parts
- We discussed the possibilities of different promoters and Team IOANNINA suggested that we use a medium strength promoter(BBa_J23111) for mf-Lon and araBAD(Arabinose inducible promoter) for bpDNase1.
- Conclusion: Team IOANNINA made the aforementioned parts compatible with the assembly standards used by them and ordered the parts from IDT and Twist.
Both the teams came to a conclusion that this circuit design checks the functionality and activity of bpDNase1 and mf-Lon.
Team IOANNINA ordered the parts to test the toxin-antitoxin system, however, they couldn't complete the experiments until the wiki freeze. Another factor adding to the delay was that the parts ordered by Team IOANNINA arrived at different times and some also got delayed due to issues at customs.
Communication - Biosafety and more
To collaborate beyond the scope of the modules of our project, both teams wanted to explore the possibility of collaborating on the part of human practices.
We collaborated on recording an episode for our team’s podcast, SynTrack (hosted on Spotify), on the topic “Biosafety and environmental impact of synthetic biology and importance of kill switches”.
Konstantinos and Shreyas recorded the podcast and had a blast and laughed at our silly mistakes while rerecording multiple takes of our dialogues.
To address the biosafety issues of our projects , we tried to contact Biosafety experts from Greece and India to get their feedback and advice. Aslaha, Shubhra from Team IISER-Tirupati_India and Elisavet, Foteini and Chrysoula from Team IOANNINA made constant efforts for this part of the collaboration. Moreover, we learnt new things about biosafety and its environmental impact while our discussions and brainstorming.
Introduction in Modelling
Team IOANNINA was facing trouble integrating their wetlab and drylab modules, Lochan and Shivam from Team IISER-Tirupati_India guided Christos, from Team IOANNINA, to build the basis of their mathematical model, to understand their difficulties and suggested them what parts of their project can be modelled or simulated, how to go about it and helped them set priorities because it was already quite late in the iGEM season.
Soon the dry lab team of Team IOANNINA came up with a kinetic model that suited their project's goals
Our own Collaboration Calendar
The calendar is a brief, yet comprehensive presentation of this partnership. This calendar was maintained by Kyrania and Shreyas once decided to take up this partnership to keep everything on track and upto date.
25/06/2021
First meeting - Idea exchange - Discussion on the general aspects of both projects
30/07/2021
Second meeting -Brainstorming ideas for Collab and Whatsapp group formation, Decided that we’d share a kill switch module with Team IOANNINA and go ahead for a long term collaboration.
06/08/2021
Human Practices team from both teams met for the first time and discussed the important aspects of human practices of both the teams.
10/08/2021
Discussion on specific parts of each project:
Thoughts on the IOANNINA team using Team IISER-Tirupati_India’s kill switch. Need from IOANNINA for a kill switch mechanism that is controlled by their designed repressors’ system, need from IISER-Tirupati_India for use of kill switch in E.coli.
Points of concern:
- Could IOANNINA test this kill switch in Bacillus, too?
- How could we control the amounts of mf-lon produced in order for the bacteria to be alive?
- Which experiments could be done? Ideas: count OD of bacteria to see if mf-lon functions correctly
20/08/2021
How can we check that mf-Lon and bpDNase1 interact?
Ideas :
- Check with HPLC or IMAC on protein level
- Check for ideal protein production by measuring bacteria OD by using different strength promoters for these 2 proteins.
21/08/2021
Deciding the topic and the script for the podcast episode.
28/08/2021
Finalised that the toxin-antitoxin system will be shared by both teams.
Team IISER Tirupati shall share the parts and sequences.
Points of concern discussed:
- Amplification of toxin gene construct in E.coli : How can we ensure that toxin is not expressed before the antitoxin is expressed in the needed concentration?
- Proposed idea: Use 2 plasmid constructs, first transform cells with antitoxin, afterwards with toxin gene
- Strength of promoter for antitoxin
- Assembly for promoters and genes (Team Ioannina)
Dry lab teams meet on 2nd Sept
HP teams to meet soon
Podcast meet to be scheduled
31/08/2021
Drylab teams met
Team IISER Tirupati suggested starting looking into their 2020 team’s wiki and other wikis for reference
IOANNINA can model the following in the given priority list
- Enzyme degradation
- Kill switch (shared module)
- HGT
Teams will meet again to discuss updates and look into the modelling prospects
01/09/2021
Human Practices meet:
Teams brainstormed new ideas for a HP collab and came up with an idea to contact Biosafety experts from Greece and India to get their feedback and advice on our projects and our shared kill switch module.
02/09/2021
Team IOANNINA updated their plan for the experiment with toxin-antitoxin.
- They’re planning to use an arabinose inducible promoter with the toxin to have control over it. This is a solution given to problem 1 from 28/08 instead of the proposal for 2 plasmids, as that would be too complicated. IOANNINA decided to assemble all the genes in one plasmid construct to reduce possibilities of transformation errors experimentally.
- Thoughts on using different promoters for the mf-lon gene. Concluded that only a medium strength promoter is enough to check the system
Plans to move further with the experiment were discussed
03/09/2021
Human Practices meet:
Continuing the idea of contacting experts, we drafted emails together and Team IOANNINA sent emails to experts from Greece while Team IISER Tirupati contacted some experts from India
05/09/2021
Team leaders of both teams and Dr Raju Mukherjee, Primary PI of IISER Tirupati signed the letter of agreement
22/09/2021
Podcast recording by Shreyas and Konstantinos
25/09/2021
Order for all relevant parts was placed
bp1DNase as 1 Gblock, and mf-Lon as 2 different sequences because of repetitive sequences in the mf-Lon gene. The araC gene was also ordered.
03/10/2021
Most team members from both teams met, have a fun chat, wiki troubleshooting, screenshots
10/10/2021
Wiki troubleshooting continues.
Ideas on how to present our partnership and our long-term communication
Ending note
With Team IOANNINA, we bonded from the beginning and over time we realised how crucial this interaction was, to provide us a better perspective on important aspects of our project. This partnership indeed shaped projects of both the teams in a positive way. We loved the way Team IOANNINA was always ready to work harder every time something new came up and we always tried our best to do the same. This journey of our long term collaboration was even though focused a lot on the wetlab, that didn’t stop us from exploring other avenues of our projects and surfing through the waves of team spirit.
We wish them all the best for their future endeavors and success in the future!
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