User:Eric M. Walters/Notebook/Spring 2012/2012/03/20

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Plating ΔacsA::accBCDA transformation culture

  • The 2mL culture was spun down gently (3k RPM, 2:30) and plated on media A plates with 500μM (??) acrylate.
  • Used beads to spread culture for the first time. Add 5-10 beads to plate, add culture (~100 μL), shake to spread, dump beads into EtOH.
  • Placed plate in new incubator room, advised to wait 5 days for sufficient growth.
  • NOTE: Should have done a no DNA MCR culture. Expecting 1-10-7 spontaneous revertants, but it would have been dandy to have a real number.

Beginning IPTG-inducible YFP project

  • Goal is to establish an IPTG-inducible strain for lab use.
  • What we have: E. coli/7002-propagatable plasmid (name??) with homology to neutral site (NS) in pAQ1 flanking (some chump promoter??), YFP, and KnR.
  • What we need: That promoter (flanked by EcoRI, NcoI (5'→3')) to be replaced with an IPTG-inducible promoter.
  • The plasmid doesn't have lacI, so will need to insert:
    • lacI under some constitutive promoter
      • The Silver lab has made a similar construct and used the native PlacI
    • lacO, the site where with IPTG derepression transcription will initiate
    • (Doesn't need to be inserted but) keep the ability to clone anything else downstream of lacO
  • I want to get the above from something out of the iGEM distribution, as it should likely exist in one part and simplify things.


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