IGEM:Harvard/2007/Meetings/Week 7
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Perry's presentation, 7-30-07 (Powerpoint 2007)
Perry's presentation 7-30-07, compatible with older versions of Powerpoint
Perry's drop experiment animation (Powerpoint 2007)
Perry's drop experiment animation, compatible with older versions of Powerpoint
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, pR also controlled by GFP
- 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)
- Drop Experiment
- 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)
- Switched fluorophores
- Shaunak
- Refer to ppt
- Sammy
- Refer to ppt
- Preliminary results look good, ie results from two-step PCR and gel are as expected
