IGEM:Harvard/2007/Meetings/Week 6

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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
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