Jordan

• Did more research into periplasm fractioning
  • In order to see if the protocol works, need JC8031 with ClyA culture, a JC8031 DeLisa cell line control (same cells, no GFP) and a cytoplasmic GFP control (if there is GFP in the fraction, full cell lysis happened and that’s bad)
  • Plan:
    • Grow up overnight culture of GFP device from interlab kit
    • Grow all three cultures in 25 mL LB cultures with Cam until OD=0.5 per DeLisa paper
    • Induce JC8301 cells with .2% arabinose (weight/final volume)
    • Grow for 6 hours at 37
    • Use cold osmotic shock protocol from Biefeld iGEM team on the drive, BUT use ice cold water instead of cell fraction buffer 2 because Kelly says Triton and SDS can interfere with protein function
    • Measure fractions on plate reader, use wavelength from interlab study
  • Need: to make cell fraction buffer (need Tris) autoclave media?, grow up cytoplasm GFP, did Paul make some arabinose?
• Made some 3M sodium acetate solution to try ethanol precipitation—Kelly says it’s worth a try—see Paul for final pH
• Ethanol precipitation protocol

Paul

• Looked into Cas9 expression cell lines with Kelly
• Cas9 should be constitutively expressed in any line without TetR repressor (so our cell line)
• Gibson assemble gRNA into mRFP reporter plasmid pSB1T3
  • Used Bradley’s Clone Kit Excel calculator
    
  • Pos. Gibson Control
  • Neg Gibson Control (no insert)
  • 10 uL rxns:
    • 4.15 uL nfH20
    • 0.41 uL 63 ng/uL mRFP backbone
    • 0.45 uL 10 ng/uL gRNA gBlock insert
    • 5 uL Gibson Mastermix
• Note for 8/24: L-arabinose 0.2% final w/v for induction of ClyA GFP