Team:IOANNINA/Partnership
One of the main aspects of our project design was ensuring that our final proposal would be as safe as possible. We strived to apply to our synthetic strain biosafety measures in order to take our design one step closer to a possible application. Thus, one of our main goals was to add to our bacterial strain a kill switch mechanism which is one of the most common mechanisms in Synthetic Biology of rendering a synthetic strain safe. The kill switch would be activated when our bacterial strain had fulfilled its role of decontaminating its substrate from tetracyclines and macrolides. While we had already designed our antibiotic sensor and the deactivation module, we were searching for a possible kill switch that would be based on a simple and effective mechanism. This is why we decided to partner with IISER-Tirupati. Team IIISER-Tirupati had already designed several kill switch mechanisms that were applied in their project but were not yet all tested in E. coli strains. After long discussions, a potential Wet Lab partnership emerged. We deemed that it would be interesting to apply their designed toxin-antitoxin system, in a different frame, specifically under the control of our antibiotic sensor system. Given IISER-Tirupati team’s previous experience in the iGEM Competition and our communication on a weekly basis, we decided to partner also in the Human Practices and Dry Lab sections. Therefore, the interaction between our teams expanded to modelling and Dry Lab, also to Biosafety issues. This whole partnership started with the need from both sides to test and apply the kill switch mechanism that was originally designed by team IISER-Tirupati and then modified in its upstream activation and deactivation regulation by our team to match the needs of our general project design.
Our discussions started in June 2021 when both teams exchanged the general concepts of their projects and brainstormed on possible ways to match our projects since we had similar goals. In July, and since our communication was already established, together we decided to proceed with this partnership, on the wet lab part. Since the time frame was very strict, we tried to arrange as many meetings as possible in a short time to come up with the best solution for our partnership. In the beginning of August, we built up the first idea, that our team would use the kill switch mechanism that has the protein toxin – antitoxin system of bp1DNase as a toxin and mf-Lon as the antitoxin. We started designing the possible experiments that could be done to test if this system could work in E. coli strains. As it would be too complicated to test the toxin-antitoxin system directly in our strain with all the three modules designed and due to lack of time for this, we decided to start with a simpler experimental design. We decided to check first if the antitoxin bp1DNase that was designed by IISER-Tirupati to destruct bacterial strains that express it could be regulated by mf-Lon protease.
The main problem that occurred on this initial thought and experimental design was how we could control the bacteria would survive if the bp1DNase was expressed earlier or in higher concentrations than the mf-Lon antitoxin protein. This would not allow us to check the interaction of these two proteins as our strains would be destructed before mf-Lon had the chance to act as an antitoxin for the system. To overcome this problem our first troubleshooting solution was to add different strength promoters to bp1DNase and mf-Lon in order to make sure that mf-Lon could repress the death of our strain that could be caused by bp1DNase overexpression. The two gene cassettes would be cloned into one plasmid carrier and the bacteria that would be transformed with this plasmid would express the two genes in different rhythms. However, by cloning both genes in one plasmid would not give us the opportunity to check at first the viability of our strain only when one of the two genes were expressed. For this reason, we thought of using two plasmid vectors, one for each gene. These two plasmids would be transformed to our bacterial strains one after another, giving our strain the opportunity to express mf-Lon in the necessary concentrations to repress the bacterial destruction caused by bp1DNase. Using two different plasmid vectors proved to be complicated as the two plasmids could possibly antagonize each other for the bacteria’s dividing mechanisms. This is why we decided to try and find a more stable solution to express the two genes in our strains for this testing experimental design. We concluded that the best solution would be to add the bp1DNase gene under the control of an inducible promoter. In this way, we could add both genes in one plasmid vector, thus minimizing the transformation steps and making our system more stable and also, we would ensure that mf-Lon would have enough time to be expressed, as we would be able to regulate when the bp1DNase would be expressed. One of the most common inducible promoters is araBAD, which is regulated by the present or absence of arabinose in the growth medium. Once we decided upon the final structure of our gene cassettes, we were ready to proceed with the design of each part. We concluded on the method that would be used to clone the gene cassettes into the vector and designed the specific DNA sequences that needed to be ordered in order to proceed with the experiments. In this process, we also designed the gene cassettes in order to test this toxin-antitoxin system in our strain that would have all three modules of our project: antibiotic sensor-antibiotic deactivation-kill switch. Due to the complexity of the sequences that needed to be ordered and the troubleshooting process that we spent a long period on, we were not able to check in the lab this precisely designed experimental procedure until the Wiki Freeze day. However, as it is an aspect of our project with great importance, we will try to test the system in the remaining time period. The partnership in Human Practices included a podcast recorded by Shreyas (IISER-Tirupati) and Konstantinos (IOANNINA). IISER-Tirupati hosted a series of podcasts (SynTrack) and we gladly took part in one of them, about biosafety, the environmental impact of synthetic biology and the importance of kill switches while dealing with GMOs. More specifically, the topics discussed in the podcast were the importance of biosafety in GMOs and their regulated environmental release, their impact in the environment, what the kill switches are, how they are regulated and the example of Deadman’s kill switch. In terms of biosafety, both Human Practices teams decided to contact Biosafety experts from Greece and India respectively to get their feedback and advice on the projects and the shared kill switch module. Although we drafted emails together and sent them to experts, unfortunately we did not get any response. 
Partnership
Overview-How it all started
Kill switch – Our Wet Lab Design
June-July 2021
August
Experimental Design
Problem
Troubleshooting
Scenario 1
Scenario 2
Scenario 3
September
October
Communication – Biosafety and more