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From 2013.igem.org

We got two plasmids from the Fussenegger group at the ETH Zurich containing a codon optimized version of bpsA

(pMM64) and svp (pMM65), respectively, for expression in HEK 297-3 cells [Muller 2012]. The first goal is to

transform the plasmids into competent E. coli (Rosetta) and check functionality of the plasmids in our cells.

Indigoidine production with pMM-plasmids I (Ilia)

After Transformation cells are grown first on Amp medium and thereafter on Kan medium.

• transform competent Rosetta with 225 ng pMM065 and 253.5 ng pMM064
• plate on Amp plate
• pick colonies from Amp plate, make liquid cultures in LB + Kan + IPTG(1 mM)
• Evening: no growth in liquid culture => prepare ON culture in LB+Amp
• Put ON culture in incubator
• prepare competent Rosetta-pMM064 from ON culture
• transform with 225 ng pMM065
• plate on Kan+IPTG plate
• no growth on Kan plate

Results and Discussion

There were no blue colonies. Transformation will be repeated and medium will be provided with both antibiotics.