IGEM:Harvard/2007/Meetings/Week 5

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

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