IGEM:Harvard/2007/Meetings/Week 6

Stephanie's Powerpoint

S.Lo's Notes

• Quorum
  • Switch fluorophores on constructs
  • Actually do dilution/extinction for fluorescence ...
  • Make graphs without normalizing for autofluorescence?
  • Keep fluorescence in one assay, run OD in parallel (unfortunate, but probably more accurate)
  • Make graphs with controls / noninduced
  • Watch RFP carefully ... try to 'bridge' these past experiments ... keep some controls/things constant
  • Alain - be careful about such long Vacufuges ...
• Fec
  • Use Quorum constructs for positive control (the ~3000 RFU) to see full range
    • In a way, this also calibrates all our experiments
  • Use E0240 (not that sketchy, apparently) for GFP fluorescence next ligation
  • PennState working on pdz and nickel domains
• MACS
  • AIDA-1 construct: MACS turned out well, about 200 colonies (versus 5 RFP colonies); assay worked well
  • Use AIDA to construct libraries
  • FACS results
    • Still haven't grown that well
  • Compatible with standard biobrick restriction sites
  • Have varied amount of antibody added (except for AIDA - exact protocol, except addition of 10 minutes to incubation, about 5uL of extra antibody each, repeat of second wash step ... first time worked; this protocol is promising then)
• Sammy
  • Recieved Primers for 2-step PCR
    • Have two products: first part of OmpA, second part: BB cutting site, strep and his at beginning as positive control, or random 10mer or 15mer (haven't gotten the randomers yet)
  • PCR for strep and his = good results
  • Cut first product with Spe1 and second with Xba, ligate
    • Ligation yielded low DNA concentrations this past weekend
    • Instead, have transformation inside protocol to increase DNA product
  • Looked at diversity - 15 and 10mers looked similar, compared through sequencing and BLAST - not much diversity?
    • Mike - the design of primers - accidental match to end of OmpA; two Biobrick sites flanking randomer site ... actually chopping randomer before ligation
      • Need to redesign primers (and more double-checking before ordering of primers)
• Target?
  • Alain suggests Calmodulin - already known binding sites; works only in presence of calcium; already have beads; well-characterized, structure known - seems like the perfect protein - believes we have antibodies, all that we need except library
• Alex
  • Used different ligation strategy
  • Every step - addition of ethanol and things ... extractions ... keep volume down and DNA concentrated?
  • Transformation - results from chemically competent cells about same as before (30 colonies per plate)
  • OneStep Electrocompetent cells (from freezer) gave only 2 colonies
  • Arsenic-sensitive promoters
    • Split normal Fec promoter into 2 different FecI (sigma factor)
    • Use directed mutagenesis to knock binding affinity