Our collaboration with team Thessaly started early in the summer when they informed us about their intention to improve part BBa_K22030059 which was introduced by a past EPFL team. Immediately, we sent them E.coli M15 strain cells with pQE-30 T7 RNAP plasmid. We also gave them our protocols for cell lysis and transformation to competent cell so they could compare them with the ones they had already As we looked into their project we realised that the methods they implement are quite similar to ours, giving us the opportunity to troubleshoot each others project. For example, we both chose RPA to amplify DNA. Both teams were struggling with extra unwanted bands on their agarose gel after electrophoresis. We ended up proposing using sequencing to determine if the correct product had been created. In October, we suggested having a skype call to check on each team’s progress and exchange a couple last-minute tips to optimize our final results. We had been having trouble analysing our gels as RPA is prone to non-specific bands when the DNA template concentration is low. To better separate the non-specific amplification bands from our amplicon, the Thessaly team suggested running our RPA product on a very high percentage gel (3-4%) at low voltage (70V) and for a long time (2h). This technique ended up being very useful to clearly differentiate between the 3 amplification product for our multiplexing experiment. We also exchanged our idea on how to design and test toeholds. Their team used 7.5 µL reactions which tends to give a more stable result than our 5 µL reactions. They also told us that adding spacers in between the T7 promoter and the toehold enhances the expression rate. On our side, we reassured Thessaly that ssDNA can also be used as a trigger for toehold regulation.