This year, our team partnered up with IISER Tirupati India 2021 team (Figure 2). Although our focus is mainly on local action, we did not want to miss out on the chance to partner up with someone living on the other side of the globe. On this page we have collected the main landmarks (Figure 1) of the ongoing relation we had established with the team IISER Tirupati India.

The Tirupati team is composed of a group of students from all over India studying in an Institute situated in the southern part. We were very amazed by the nice opportunity that the competition gave us in terms of connecting with the synbio community from all around the world.

Our captain Ivelina spotted a message in the channel 4-collaboration on the iGEM 2021 Global slack environment where Team IISER Tirupati were explaining their ideas on their project and she found out that we may have some points in common as well as some differences. This way we could both learn from each other without too much overlapping. We decided to email the team and they luckily replied and wanted to meet us!

We met for the first time with iGEM Turipati on the 16th of June (Figure 3). In that meeting some members of both teams were present and both projects were presented.

Team Tirupati presented us their project so far and we learnt really interesting things about it. Their project is aiming to create a new contraceptive, which will involve the delivery of GMO commensal bacteria into the female upper reproductive tract.

These bacteria will produce proteases once they reach the fallopian tube. These secreted proteases will affect the zona pellucida of the ovum by hardening it and therefore making it impossible for fertilization by sperm. The bacteria will have 2 kill switches - one of them will be activated by light in the event of bacteria slipping out of the reproductive system and the other is for reversibility of the contraceptive.

We were really 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 discuss about:

• Wet lab: we found out that they were also going to use S. cerevisiae, as the proteases they will work with are eukaryotic and have a large size, so they would like to confirm the heterologous production works in a eukaryotic organism as well as in bacteria. They were also going to use the Golden Gate Assembly. We also found out that they were going to engineer Bacillus subtilis, a bacteria that we also used to compare it with our chassis. Regarding this, they suggested that we look for B. subtilis strains with protease gene knockouts if we want to be able to compare the characteristics of alpha-amylase synthesised in the native organism and in our GMO.
• Dry lab: they are planning to produce their protein in an uncontrolled environment. Therefore, most of the modeling techniques we are planning on using will not be applicable to their project and vice versa. Possible collaboration could be achieved in the field of molecular dynamics. They are planning on making their protein interact with the zona pellucida in the ovum. They want to model this process using molecular dynamics. At that point we weren’t very certain of the molecule that we were going to work with. If we had started working on urease inhibitors, we would have also been using some form of molecular dynamics.
• Collaboration ideas: we were discussing the medal criteria Improvement of Existing Parts and they mentioned there are existing libraries for Bacillus subtilis and S. cerevisiae parts (in iGEM) are not well-characterised, so we could think of a way to collaborate on a part that we can improve and divide the work. They also asked us about our lab availability, because they had some restrictions in India and it is still unsure when they can start lab work. At that moment they were hoping to be able to start lab work in the middle or end of July and have at least 2 months to run experiments, but that might not be the case. 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) was discussed with Shreyas, their other co-leader.

Conclusion: They will be happy to collaborate with us on the Gold medal criteria Improvement of Existing Part (wetlab collaboration!). We can also look into whether they can 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) A dry lab collaboration will only be possible in the field of molecular dynamics, if we start working on urease inhibitors.

Overall the meeting was very smooth and both teams were very enthusiastic about helping each other. We had really nice feedback from them.

We met again on the 20-7 (Figure 4) to discuss further the wet lab part of our projects. In this meeting more members of our wetlab team joined! We heard from Tirupati that they will use two inducible promoters - GAL1 (galactose induced) and CUP1 (copper inducible promoter). They presented their yeast construct. We discussed the chassis that both teams were using and had in common, S. cerevisiae and B. subtilis. For team Tirupati S. cerevisiae was going to be employed to test the genetic construct, to make sure that the big proteases that they wanted to express were produced. B. subtilis was employed as the proof-of-concept bacteria that would deliver the proteases in the fallopian tubes. We talked about the strains that both teams were planning to use. They were planning on using Bacillus subtilis 168 strain and adding a protease inhibitor cocktails mix to the culture media. These mixes are commonly used in heterogeneous protein production in BS168 and other organisms.

Conclusions:

Several conclusions were withdrawn from this meeting. The most feasible option for a long term collaboration/partnership that we see now is 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 the other team can use the protocols from the others (as we were more experienced with S. cerevisiae and they were with B. subtilis). We learned some valuable information to use in our project. Shreyas told us about proteases inhibitors that may enhance the production of our heterologous protein. We actually did a bit further research on the subject and thought about how to implement this information into our project. We also found out about the Lucid app, that we will later use to illustrate our construct. We created a slack channel where we could share protocols, articles and doubts.This way all the members of the teams would take part in the collaboration.

We were ready for our monthly meeting, it happened on the 18th of August. In this meeting we made the decision to extend the partnership beyond the wet lab. 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 the one from the other but we wanted to still work together on troubleshooting, as it had been really useful in the past. That meant that once the Tirupati team starts with transforming the S. cerevisiae or once the Groningen team starts with growing the B. subtilis, we could set up a meeting to discuss the challenges/tips the other team has learned so far.
• Human Practices: Tirupati team is working on a podcast with other iGEM teams and is working on a survey for which they had help from their university ethics committee. We discussed that the Groningen team could help make an episode for the podcast and Tirupati team could send the Groningen team their survey/the input they got from the ethics committee. Our team worked hard on the information sheet and informed consent sheet and we sent it to the Tirupati team so they could use it a bit.
• Dry lab: we told Tirupati our plans on using the ART tool and the AlphaFold2 pipeline to have a better insight of our device. We offered our help with this tool if they were interested in any. We also heard about their modelling approach, we would perform troubleshooting sessions
• Wiki: we were happy to hear that the Tirupati team had more experience with coding, as we all needed to learn from zero. They offered their help on the matter and we actually contacted Ankush, who helped us overcome some minor bugs in our code.

During this month we kept in touch through the slack channel.

Two members of our team finally sat down with Shubhra to participate in their podcast (see Figure 6). On the episode we talked about synbio in the food industry, their potentials and also the risks. We really enjoyed the previous discussion we had with Shubhra about it and we learned a lot about the topic. If you want to know more about synbio in the food industry check Turipati's podcast on spotify!

We finally met with Shreyas (Figure 7) a couple of weeks before the wiki freeze. It was a really 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 seems 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 are really glad to have shared this exhausting but unforgettable iGEM season with team Tirupati. We are thankful for their useful insights in different aspects of the competition, from the wetlab through coding the wiki, to human practices. We wish them the best and a lot of success!