Team:Heidelberg/Templates/Indigoidine week12 overview


Indigoidine Production - indC

Since S. lavendulae DSM40708 does not carry the bpsA gene, we used indC from Photorhabdus luminescens laumondii TT01 instead (Brachmann 2012).
The PPTase Sfp, originating from the B. subtilis strain 168, has been shown to exhibit a broad substrate specificity (Nakano 1988) and thus was used to activate indC on pRB3. pRB3 is similar to previous plasmids (indigoidine synthetase and PPTase on pSB1C3) but contains a KpnI cutting site as a spacer between the first ribosome binding site (RBS) and the indC coding sequence, a BamHI cutting site as a spacer between the second RBS and the sfp coding sequence as well as a NheI cutting site at the end of sfp. Therefore, this plasmid can be used with both Gibson or CPEC and a classical restriction cloning assembly strategies.
If pRB3 is functional, we will continue testing various combinations of PPTases and indigoidine synthetases.

pRB3 is a functional pSB1C3 construct with the genotype lacI-Promoter-RBS1-KpnI-indC-RBS2-BamHI-sfp-NheI-pSB1C3ΔmRFP1. The blue phenotype of pRB3-transformed E.coli TOP10 cells proofs production of the blue pigment indigoidine after 30 hours and incubation at 37 °C. Transformed cells expressing the indigoidine synthetase gene and producing indigoidine grow much slower than usual TOP10 cells. This effect was already reported by other groups (Owen 2011) and is supposed to be due to the slight toxicity of indigoidine.
Next week we will amplify other PPTases and assemble plasmids with combinations of indC, bpsA(pMM64), sfp, svp, svp(pMM65) and entD. The ladder is the endogenous PPTase of E. coli and considered to be inefficient in activating indigoidine synthetases (Takahashi 2007). We want to test and compare entD with the other PPTases when overexpressed on a plasmid.