IGEM:Harvard/2007/Meetings/Week 5: Difference between revisions
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[[Media:sloweek5igem.ppt|Stephanie's Week 5 Powerpoint]] | [[Media:sloweek5igem.ppt|Stephanie's Week 5 Powerpoint]] <br> | ||
[[Media:gxuweek5igem.ppt|George's Week 5 Powerpoint]] | |||
<br><br> | <br><br> | ||
<b>S.Lo's Notes:</b><br> | <b>S.Lo's Notes:</b><br> | ||
<u>Quorum</u><br> | <u>Quorum, Stephanie and George</u><br> | ||
*Read literature: how much steepness do we want for quorum? (Might not be necessary given correct controls) | *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) | *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? | *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 | |||
<br> | |||
<u>Fec Project, Shaunak and Ellenor</u><br> | |||
*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 | |||
<br> | |||
<u>MACS, Kevin</u> | |||
*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? | |||
<br> | |||
<u>New Strategies: Sammy</u> | |||
*Sequential PCR | |||
*Forward primer with random-er | |||
*ligate two products | |||
*MACS worked this past spring - discuss protocol in future |
Latest revision as of 11:27, 16 July 2007
Stephanie's Week 5 Powerpoint
George's Week 5 Powerpoint
S.Lo's Notes:
Quorum, Stephanie and George
- 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
MACS, Kevin
- 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