Team:Heidelberg/Templates/Indigoidine week9 overview


Indigioidine Production – bpsA

This week we assembled our first pSB1C3 derived plasmid pKH1, which carries both the bpsA and the svp gene with the lacI-Promoter BBa_R0010 using a Gibson (Gibson 2009) and a CPEC (Quan 2009) cloning strategy. We replaced the mRFP1 coding sequence with the bpsA and svp coding sequences with the RBS B0034, respectively. Thus we assemble three fragments in total, which are pSB1C3ΔmRFP1 (Primer NI9/10), bpsA (Primer NI1/6) and svp (NI7/8).
With this plasmid it is possible to produce indigoidine in E. coli TOP10 without IPTG induction, since TOP10 cells lack the lac repressor gene.

As the PCR amplifications of all fragments for the assembly of pKH1 were positive, we could perform both the Gibson and CPEC assembly for pKH1.
After IPTG induction, co-transformation of BAP1 with the originalpMM64 and pMM65 yielded blue liquid cultures. We compared the blue color using four samples induced by different amounts of IPTG. We couldn't detect significant correlation between indigoidine production and varying IPTG concentration.
Since we have had problems with the plasmids received from Zurich and we wanted to use the original gene rather than a codon optimized version, we planned to extract the bpsA indigoidine synthetase gene directly from the organism it originates from, namely Streptomyces lavendulae subsp. lavendulae strain ATCC11924 (Takahashi 2007). As the DSMZ (German Collection of Microorganisms and Cell Cultures) doesn't provide this specific strain any longer, we used another S. lavendulae subsp. lavendulae strain (DSM40708) expecting the bpsA gene to be present as well. We cultivated S. lavendulae in both YEME medium (Kieser 2000) and GYM medium (DSMZ medium No 65) at 28 °C.