Team:Heidelberg/Templates/Indigoidine week10 overview


Indigoidine Production - bpsA

For the PCR amplification of native bpsA, Streptomyces lavendulae subsp. lavendulae DSM40708 was grown on GYM agar plates. We wanted to use the native indigoidine synthetase bpsA and the PPTase svp from pMM65 and to assemble pRB1, which is similar to pKH1. In this new assembly approach, we amplify the bpsA gene from the S. lavendulae genome instead of using a codon optimized sequence. Furthermore the svp coding sequence was placed behind a weaker promoter (BBa_B0029) since the PPTase has to activate every indigoidine synthetase only once and thus is not required in huge amounts (Lambalot 1996).
In the subsequent weeks we wanted to exchange the Thiolation-domain (T-domain) of bpsA with T-Domains of other NRPS modules and show that it can be activated by various PPTases. We wanted to find out whether there are differences in the PPTases' efficiency concerning the activation of engineered indigoidine synthetases.

The PCR amplification of bpsA was unsuccessful. We will try again with a new set of primers.
In addition we will try to amplify the native svp PPTase from Streptomyces verticillus ATCC15003, which has been described in previous studies (Sanchez 2001).