Our fellow iGEM colleagues will surely agree with us when we say that our precious bacteria are very stubborn and mischievous and that they don't always want to behave like we would want them to. It takes a group of very stubborn and cunning people to trick them into doing what we want. Last few months were a tug-of-war between experimentalists and Pavlov's coli. We definitely got pulled on several occasions. But we are a stubborn bunch of people, so let us present how we pulled back and got as far as we did.

Construction of an AHL sensor: esabox/EsaR system

During our brainstorming sessions in the months before "hitting the lab" we put special focus on trying to find a system for an AHL sensor which would be based on a repressor rather than activator. We estimated this would allow us to build a better AND gate. We found the esabox/EsaR system. Esaboxes are binding sites for EsaR. We saw a potential to create many different combinations of hybrid promoters by placing esaboxes on different places and in different numbers to the promoter region. However, due to the strong secondary structure and repetitive sequences, esaboxes proved to be a challenge to create. We tried several different approaches and at the end we managed to construct five different combinations of synthetic promoters with esaboxes in different numbers and different spacings. We additionally managed to create more than 10 different AND gate promoters with esaboxes and pNorV. In total we created almost 20 different promoters just by varying the location and number of esaboxes in the promoter region. We were able to observe what is the effect of spacers and number of esaboxes on the behavior of the promoters.

When we started with testing phase of our esabox promoters we observed that while we can successfully repress transcription with our EsaR repressor, we cannot successfully release the repressor from the promoter.

We were able to build:

• five different promoters with different combinations of esaboxes as roadblocks

• EsaR repressor which can get released from promoter upon induction

Construction of a recombinase based switch

Permanent memory is an important aspect of our project. The most obvious way of achieving permanent memory in cells is by changing their DNA. Recombinases provide an elegant way to create such uni-directional switch.They flip a sequence flanked between two recombinase recognition sites. However, it is important that we consider the dynamics of recombinases when we try to build such switch. Intuition often fails when it comes to estimating the dynamics of a design or a system. This is where a collaboration between theory and experiments plays an important role. In our project, the interaction between theory and experiments has been important since the beginning in the construction a recombinase based switch. In their quest to minimize the potential leakiness of our system, modelers discovered a better implementation of our idea. That means that after we already spent a month in the lab designing and building our system,they provided us with important simulations of our system and offered a novel design which we immediately started to build. On the other hand, modelers needed rigorous experimental data to be able to characterize recombinases and their dynamics. This would again in turn help experimentalists create a better design. In our project we studied dynamics of two different recombinases, bxb1 and tp901 by creating reporters for the respective recombinases and perform rigorous experiment about their dynamics. We studied properties of three versions with different degradation tags for each of the recombinases. We were able to show the flipping rates for different recombinases and attempted to improve them.

We were able to construct reporters for recombinases with which we were also able to show the differences in dynamics of recombinases when different degradation tags were added