Team:Caltech/Week 10

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Latest revision as of 19:19, 20 August 2010

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Monday 8/16

Group meeting!
Ran CPCR gels on L31, L34, L43, L58 & L71
- All negative.

Tuesday 8/17

Took our team photo!
Ran more CPCR of L58 & L81.
- Gel was negative.
Cultured HSP-LacI-GFP cells in LB + 1M glucose to try to up-regulate LacI repression of GFP production. Incubated cells at 37°C overnight.

Wednesday 8/18

LacI appears fully repressed in L62 cells after overnight incubation at 37°C.
- Will now spin down cells and resuspend in LB + IPTG to see if overnight incubation now activates GFP production, as predicted.
Picked more CPCR colonies of L81 & L58 (x6) to try to find plastic production cells.
Transformed L60 & L61. Plated on plates containing 50μL 50mM glucose (dried).
- We hypothesize that the reason these ligations produced no colonies is due to leaky LacI repression, allowing a low level of lysis expression, eventually killing all the cells. Will see if there are cells on these plates tomorrow.
Submitted our poster deign for printing for Sasha's SURF presentation tomorrow.
Brian illuminated half of a plate containing a lawn of HSP+GFP cells to try some rudimentary printing. No fluorescence after 3 hours; will check tomorrow.

Thursday 8/19

L60 & L61 grew colonies! Began CPCR to find correct inserts.
- Gel is negative - all bands appear to be RFP.
Gel of L58 CPCR was negative again. Will design 'ligation primers' to connect the two for L58. L81-4 was correct; miniprepped the DNA for ligation tomorrow.
Brian's test printing attempt plates appear somewhat green, but there is no boundary between the lit and unlit regions. Will re-try test more carefully next week.
Sasha presented our SURF poster today! People seem pretty excited about the project, but we need more data.

Friday 8/20

Began rough HSP activation kinetics experiment.
- With 3 cultures of HSP+GFP cells grown 2 days @ 30°C: incubated two at 42°C for {5, 10}min, leaving the third on the bench for 15 min. Took 200μL samples of each to measure the fluorescence & OD600 right after incubation and every 15 minutes thereafter for 5 hours. Incubated the tubes at 30°C while not taking aliquots.
- Plotted the fluorescence (RFU), normalized for OD (RFU/OD600) vs time.

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@@ Line 1: / Line 1: @@
 {{Caltech_iGEM_10|
 Content=
-[[Team:Caltech/Notebook|Back to Notebook]]
+{{:Team:Caltech/WeekTemplate}}
 ==Monday 8/16==
-:<math>m= \frac{\mbox{rise}}{\mbox{run}}= {\mbox{change in } y \over \mbox{change in } x} = {\Delta y \over{\Delta x}}.</math>
+* Group meeting!
+* Ran CPCR gels on L31, L34, L43, L58 & L71
+** All negative.
 ==Tuesday 8/17==
+* Took our team photo!
+* Ran more CPCR of L58 & L81.
+** Gel was negative.
+* Cultured HSP-LacI-GFP cells in LB + 1M glucose to try to up-regulate LacI repression of GFP production. Incubated cells at 37&deg;C overnight.
 ==Wednesday 8/18==
+* LacI appears fully repressed in L62 cells after overnight incubation at 37&deg;C.
+** Will now spin down cells and resuspend in LB + IPTG to see if overnight incubation now activates GFP production, as predicted.
+* Picked more CPCR colonies of L81 & L58 (x6) to try to find plastic production cells.
+* Transformed L60 & L61. Plated on plates containing 50&mu;L 50mM glucose (dried).
+** We hypothesize that the reason these ligations produced no colonies is due to leaky LacI repression, allowing a low level of lysis expression, eventually killing all the cells. Will see if there are cells on these plates tomorrow.
+* Submitted our poster deign for printing for Sasha's SURF presentation tomorrow.
+* Brian illuminated half of a plate containing a lawn of HSP+GFP cells to try some rudimentary printing. No fluorescence after 3 hours; will check tomorrow.
 ==Thursday 8/19==
+* L60 & L61 grew colonies! Began CPCR to find correct inserts.
+** Gel is negative - all bands appear to be RFP.
+* Gel of L58 CPCR was negative again. Will design 'ligation primers' to connect the two for L58. L81-4 was correct; miniprepped the DNA for ligation tomorrow.
+* Brian's test printing attempt plates appear somewhat green, but there is no boundary between the lit and unlit regions. Will re-try test more carefully next week.
+* Sasha presented our SURF poster today! People seem pretty excited about the project, but we need more data.
 ==Friday 8/20==
+* Began rough HSP activation kinetics experiment.
+** With 3 cultures of HSP+GFP cells grown 2 days @ 30&deg;C: incubated two at 42&deg;C for {5, 10}min, leaving the third on the bench for 15 min. Took 200&mu;L samples of each to measure the fluorescence & OD600 right after incubation and every 15 minutes thereafter for 5 hours. Incubated the tubes at 30&deg;C while not taking aliquots.
+** Plotted the fluorescence (RFU), normalized for OD (RFU/OD600) vs time.
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