IGEM:Harvard/2007/Meetings/Week 5

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Stephanie's Week 5 Powerpoint
George's Week 5 Powerpoint

S.Lo's Notes:
Quorum, Stephanie and George

  • Read literature: how much steepness do we want for quorum? (Might not be necessary given correct controls)
  • Use ridiculous wavelength for light scattering / cell growth (test on non-fluorescent constructs to ensure this works)
  • How to ensure J-T is not self-induction?
    • Fluorescent microscope
    • Knock-out OHHL from one construct ...? Figure this out ... schematic ...
  • Wash during/with dilution
  • Make plots from now on as RFU versus cell number rather than time

Fec Project, Shaunak and Ellenor

  • Successful site-directed mutagenesis to remove Pst/Spe sites
  • Chose 4 targets
  • Grew up bacteria from Germany: AA93 (Fec knockout) and two other types
  • No growth from cotransformation (flawed protocol?)
    • Low probability of both plasmids uptaken
    • Use more sample
  • Figure out vector these are in
  • Mutagenesis for library?
  • Electroporation of cells that already have plasmid

MACS, Kevin

  • OmpA1 ran through 3 different assays and one FACS
    • Unsucessful; OmpA1 likely less robust as we had originaly thought
    • Exact reasons not clear yet
  • His/strep tag attached to AIDA-1 instead - perhaps will express better results as surface expression protein
  • Try Sammy's strategy: insert tags to loop region of OmpA1 construct
  • No solid evidence that membrane proteins are binding well
  • Perhaps - obtain constructs from literature and see if these work as claimed?
    • Just need to find one strategy that works
  • Can be tied to Fec system - if loop region is changed, might as wel use it for targeting as well as signaling (simultaneously?)
  • Dilute our cells down: be more conscious about fluorescent antibodies - we had used a lot of cells
    • Dilute samples largely; more antibodies per cell; FACS and MACS again
  • Elongate incubation with antibodies: our 10 minutes were probably too short
  • Detection limit: Call company to ask?

New Strategies: Sammy

  • Sequential PCR
  • Forward primer with random-er
  • ligate two products
  • MACS worked this past spring - discuss protocol in future