Team:Heidelberg/Templates/Indigoidine week16 overview


T-Domain Shuffling – PPTase Plasmids

This week we wanted to prepare plasmids for the T-Domain exchange experiments. We assembled four plasmids pRB15-18, each containing one PPTase on the pSB3K3-backbone with a kanamycine resistance. These plasmids should be used for co-transformations with the engineered indigoidine synthetase indC on a pSB1C3 plasmid pRB19 with a chloramphenicole resistance.

We successfully assembled pRB15-18.

BioBricks for the Registry

As a first set of BioBricks we chose the coding sequences of the four PPTases sfp, svp, entD and delC as well as the indigoidine synthetase indC. Since indC contains two internal RFC[10] cutting sites (EcoRI and SpeI), the DNA sequence had to be mutated for the part submission. Therefore we first assembled pRB21, which is identical to pRB3 but doesn't contain a PPTase.

We successfully assembled pRB21 and amplified the PPTases with the BioBrick RFC[10] prefix and suffix (Primer KH13-20). Next week we cloned the PPTases into the pSB1C3 backbone using restriction cloning and removed the EcoRI and SpeI cutting sites contained in indC using a CPEC mutagenesis strategy.