IGEM:Harvard/2007/Meetings/Week 7

[[Media:Perry Tsai presentation 7-30-07.ppt | Perry's presentation, 7-30-07 (Powerpoint 2007)]]

[[Media:Perry Tsai presentation 7-30-07 (older version compatible).ppt | Perry's presentation 7-30-07, compatible with older versions of Powerpoint]]

[[Media:Drop experiment.ppt | Perry's drop experiment animation (Powerpoint 2007)]]

[[Media:Drop experiment (older version compatible).ppt | Perry's drop experiment animation, compatible with older versions of Powerpoint]]

[[Media:sloslide0730.jpg | Stephanie's slide]]

[[Media:New_grp_presentation.ppt | Sambu's ppt (Powerpoint 2007)]]

[[Media:George Xu presentaiton 7-30-07.ppt | George's presentation, 7-30-07]]

S.Lo's Notes
 * Perry
 * GFP-mek/PDZ cell binding assay: No significant fluorescence
 * In vitro assay
 * Glutathione bead used
 * Scale of original invitro assay not great (485/538, quorum-sensing protocol)
 * Results not clear when using different pair of wavelengths (for EGFP)
 * Quorum
 * Drop Experiment
 * Plate Receiver, lawns
 * Add Red OHHL sender into middle of agar plate
 * Green halo seen around center
 * Rebuilding Voigt construct
 * lux R and lux I controlled by lux pR promoters, GFP also controlled by pR promoter
 * Perry building through PCR/digest assembly
 * Worked until attempt to put together luxR-luxpR promoter and B0034-luxI - 2kb fragment disappeared - perhaps forward primer just binds to luxI and amplifies just luxI end part
 * Question: why need to rebuild this construct? Answer: more "offness" of construct
 * Alain's comment: Voigt paper has luxI and luxR in opposite directions; play with Biobrick sites and make it backwards
 * Harris - ask Voigt for construct? (George will email)
 * George
 * Switched fluorophores
 * GFP/RFP folding/maturation assay?
 * Growth, not induction, of T02 (small range of GFP, comparable to tetR GFP sender)
 * FACS
 * Overnight cultures, serially diluted (1:10, 1:100, 1:1000, 1:10000)
 * Measured OD before appt (only grew up for a few hrs)
 * Wanted to see lower concentrations (below quorum) w/ mean GFP per OD lower than quorum-reaching dilutions
 * Redo with JT diluted multiple times, watch percent of total cells fluorescent
 * Go to OD that's higher (compare/contrast quorum-reaching)
 * Shaunak
 * Refer to ppt
 * Sammy
 * Refer to ppt
 * Preliminary results look good, ie results from two-step PCR and gel are as expected
 * Kevin
 * Refer to "Plates" subpage of Wiki; colony counts of MACS
 * Allow selection of white colonies, only a few red
 * Flow cytometry: hard to find difference between negative and positive
 * Second time - red, no white, colonies
 * Fluorescent tags not compatible with wavelength?
 * Outlook: Run another MACS this week, try to get selection
 * Use a background that is not fluorescent (lacZ?)
 * Try MACS, purifying, then going to FACS
 * Focus on inserting library
 * Alex
 * Miniprep did not work