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From 2013.igem.org

Transformed Rosetta with pMM64 and pMM65 didn't show a blue phenotype. Transformation will be repeated.

Indigoidine production with pMM-plasmids II (Ilia)

• one blue colony on plate
• make ON culture in LB + Amp + Kan + IPTG(1 mM)
• transform BAP1 with 169 ng pMM064, plate on LB+Amp+IPTG
• ON culture opaque, but colorless
• plate contains BAP1-pMM064 colonies, but colorless
• prepare fresh IPTG
• inoculate 5 ml LB + Amp + Kan + IPTG(10 mM) with 1 ml from Rosetta-pMM064-pMM065 ON culture
• inoculate 5 ml LB + Amp + IPTG(10 mM) with BAP1-pMM064
• ON cultures opaque, but colorless
• according to <bib id="pmid23053349"/>, indigoidine is extremely unstable, they did not observe any color above

30°C

• inoculate 5 ml LB + Amp + Kan with 1 ml from Rosetta-pMM064-pMM065 ON culture
• inoculate 5 ml LB + Amp with 1 ml from BAP1-pMM064 ON culture
• when cultures reach opaqueness: add 1 mM IPTG, shake at 25-28°C
• liquid cultures opaque, but colorless
• indigoidine extremely unstable, but incubator has transparent door, incubator room well lit
• prepare Amp+IPTG plate, plate BAP1-pMM064 from ON culture, leave at RT in drawer -> dark
• inoculate 5 ml LB + Amp + Kan with 100 µl from Rosetta-pMM064-pMM065 ON culture
• inoculate 5 ml LB + Amp with 100 µl from BAP1-pMM064 ON culture
• when cultures reach opaqueness: add 1mM IPTG, wrap in tin foil (-> dark), shake at 25-28°C
• no coloration of liquid cultures
• white colonies on plate

Results and Discussion

Cells don't show a blue phenotype. We received the E. coli BAP1-strain which is commonly used for heterologuos

expression of NRPS. It carries sfp, a 4'-Phosphopanthetheinyl-Transferase from Bacillus subtilis to be able to

activate NRPS PCP-domains (i.e. T-Domains)[Pfeifer 2001]. Transformation with pMM64 (bpsA) should result in

indigoidine production since sfp was previously shown to activate indigoidine synthetases [Yu 2013].