IGEM:Harvard/2007/Meetings/Week 5

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