Team:Heidelberg/Templates/Indigoidine week7

From 2013.igem.org

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].

Indigoidine production with pMM-plasmids III (Ilia)

  • co-transform BAP1 with 84.5 ng pMM64 and 225 ng pMM65, plate on Amp + Kan + IPTG

plate

  • inoculate 4 ml LB + Amp with BAP1-pMM64, grow at 37°C
Gel electrophoresis of the pMM64-pMM65 digestion. Lane 1: NEB 2-log ladder; lane 2: Undigested pMM64; lane 3: Digestion of pMM64 with EcoRI+NotI; lane 4: Digestion of pMM65 with Pst+XhoI
  • only white colonies on Amp + Kan + IPTG plate
  • make miniPrep of BAP1-pMM64 ON culture -> 19 ng / µl
  • digest 190 ng pMM64 with EcoRI+NotI (10 µl of miniPrep, 30 µl total volume), digest 450 ng pMM65 with PstI+XhoI

(30 µl total volume) => expect: 2 bands at 3.5 and 4 kb for pMM64, bands at 3.3 and 0.8 kb for pMM65

  • pMM64 shows fragments at 4 kb and 5 kb => wrong plasmid
  • pMM64 shows one weak band at 4 kb => might be right, but NanoDrop concentration measurement wrong
  • add 30 µl H2O to original Fussenegger filter paper of pMM64
  • transform BAP1 with 2 µl pMM64 from original filter paper, plate on Amp + IPTG, grow

at 37°C

  • transform TOP10 with 2 µl pMM64 from original filter paper, plate on Amp, grow at

37°C

Gel electrophoresis of the pMM64 digestion. Lane 1: NEB 2-log ladder; lane 2: Undigested pMM64; lane 3: Digestion of pMM64 with EcoRI+NotI
  • White BAP1-pMM64 colonies
  • inoculate 4 ml LB + Amp with TOP10-pMM64 from plate
  • miniPrep: 15.2 ng / µl in 27.5 µl
  • digest 152 ng pMM64 (10 µl of miniPrep) with EcoRI+NotI (total volume 30 µl)
  • 2 bands at 4.5 and 6 kb => wrong plasmid
Gel electrophoresis of the pMM64-pMM65 digestion. Lane 1: NEB 2-log ladder; lane 2: Digestion of pMM64 with EcoRI+HindIII; lane 3: Digestion of pMM65 with EcoRI+HindIII
  • inoculate 4 ml LB + Amp with TOP10-pMM64 from plate
  • miniPrep: 14.6 ng / µl in 27.5 µl
  • digest 175.2 ng (12 µl from miniPrep) pMM64 with EcoRI+HindIII (30 µl total volume, NEB buffer 2 + BSA)
  • digest 7 µl pMM65 with EcoRI+HindIII (30 µl total volume, NEB buffer 2 + BSA)
  • pMM64: 2 bands at 4.5 and 6 kb => wrong plasmid
  • pMM65: 1 band at 4 kb => might be right
  • add 30 µl H2O to original Fussenegger filter paper of pMM65
  • transform TOP10 with 10 µl pMM65 from original filter paper, plate on Kan, grow at

37°C

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