IGEM:Harvard/2007/Meetings/Week 7: Difference between revisions
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[[Media:sloslide0730.jpg | Stephanie's slide]] | [[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]] | |||
<br><br> | <br><br> | ||
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****Green halo seen around center | ****Green halo seen around center | ||
***Rebuilding Voigt construct | ***Rebuilding Voigt construct | ||
****lux R and lux I controlled by lux pR promoters, | ****lux R and lux I controlled by lux pR promoters, GFP also controlled by pR promoter | ||
****Perry building through PCR/digest assembly | ****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 | *****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 | ||
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**Switched fluorophores | **Switched fluorophores | ||
***GFP/RFP folding/maturation assay? | ***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 |
Latest revision as of 08:48, 30 July 2007
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
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)
- 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
- 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