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From 2013.igem.org

Indigoidine Production – Testing PPTases

pRB3 contains indC and sfp and results in a blue phenotype after tranformation of E. coli MG1655, BAP1 and TOP10. pRB4-10 plasmids code for different combinations of either indigoidine synthetase indC or bpsA and the PPTase sfp, svp, svp (pMM65) or entD. Transformation of E. coli TOP10 showed that all combinations result in indigoidine production. This week we validated these plasmids using colony PCR screenings and Sanger sequencing (GATC-biotech, Konstanz).

The PCR screening was inconclusive but sequencing results showed that all combinations of indC or bpsA with sfp, svp, svp (pMM65) or entD have been assembled correctly.
These results are remarkable since entD was previously described as an inefficient activator of indigoidine synthetases (Takahashi 2007). In the following week we prepared separate PPTase plasmids for co-transformation with engineered versions of indC. Also, we included the delC PPTase from D. acidovorans SPH-1, which is the PPTase involved in delftibactin synthesis.