IGEM:Harvard/2007/Meetings/Week 5

[[Media:sloweek5igem.ppt|Stephanie's Week 5 Powerpoint]] [[Media:gxuweek5igem.ppt|George's Week 5 Powerpoint]] S.Lo's Notes: Quorum, Stephanie and George Fec Project, Shaunak and Ellenor MACS, Kevin New Strategies: Sammy
 * 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
 * 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?
 * Sequential PCR
 * Forward primer with random-er
 * ligate two products
 * MACS worked this past spring - discuss protocol in future