- 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
- 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