Week 1

At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was done earlier that week with the Tripeptide I - NRPS. After a second round of picking and restriction digest, positive colonies were also found for the missing constructs and DNA was sent to sequencing. If the sequencing turns out to be correct, we can transform BAP I cells with the missing constructs, and can either way start with the preparation for mass-spec. Besides that, primers for the Tyrocidine-Indigoidine-Fusion-Project were designed and ordered.