IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 4: Difference between revisions

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*[http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]
====Plate Reader ALL 96 WELLS BABY!====
====Plate Reader ALL 96 WELLS BABY!====
Stephanie set up an overnight fluorescence plate reader test with dilutions of cells instead of swabs in order to keep the number of cells more consistent between the plates. Several things were being tested on this plate. Read the entry for more information:
[[Shlo/notebook#7.2F13|Stephanie's notebook entry]]

Revision as of 10:56, 13 July 2007

7/09/07

Colony PCR's

George, Stephanie, and Perry ran colony PCR's on the constructs that Perry and Stephanie transformed on the previous Friday.

The following colonies turned out to be correct:

Stephanie's notebook entry

7/10/07

Plate Reader

Stephanie made 1:20 dilutions into 200 and grew samples of T9002 (both {incubated overnight with 100nM OHHL} and {incubated overnight without induction}),

Stephanie's notebook entry

Unfortunately, the results/data for this experiment were lost due to Windows automatically restarting itself and not saving any data.

More Colony PCR's

Stephanie, Perry, and George ran more colony PCR's. The parts were split up into three gels, with different extension times for each gel group:

Results: The following parts ended up being correct:

Stephanie's notebook entry

07/11/07

Setting Up the Overnight Fluorescent Plate Reader

Stephanie set up the plate reader for an overnight plate reader experiment with:

All the above samples were 200 µL cultures of 1:100 dilutions of overnight liquid cultures. In addition, Stephanie ran six samples of the J23039 > T9002 that were prepared by dipping a colony swab into 200 µL of LB medium.

Stephanie's notebook entry

FACS setup

The following parts were taken from overnight cultures, diluted 1:20, grown for one hour (at which point they reached the indicated OD's), and then induced with 100 nM OHHL. The original plan was to spin down and resuspend in PBA at two hours and four hours after induction. However, we forgot to put the cells in the incubator after the two hour sample, so we only had the two hour sample. The parts:

Sample OD at induction
T9002 (+) not used
T9002 (-) 0.379
I13273 0.430
F2620 > E0240 0.527
J23039>T9002 0.317
B0015 0.496
I13522 0.397
I15311 0.482
S03608 > I13507 mixed with T9002 0.427
S03608 > I13507 mixed with I13273 0.395

The T9002 (+) was an overnight culture that was induced the day before. According to Stephanie, "Interestingly, the T9002 (+) had relatively little fluorescence compared to the constitutve samples. Perhaps the fluorescence had started to decrease over time, or perhaps the T9002 was not activated that strongly ... this is something we can measure over time with the plate reader ... if it does not turn off again and lose our data."

Stephanie's notebook entry

FACS result

Stephanie's notebook entry

Sequencing

George sent out the following for Genewiz sequencing:

George's notebook entry

7/12/04

Miniprepping

Stephanie miniprepped the liquid cultures that were grown on 7/11/07 in preparation for building the constitutive tetR expresser. The parts:

Stephanie's notebook entry

Colony PCR

Stephanie and George colony PCR'ed some parts:

Stephanie's notebook entry

Sequencing Results

The results for the Genewiz sequencing from yesterday came back:

Media:071107sequences.zip

Digests

George performed the following digests in preparation for building the constitutive tetR generating construct:

George's notebook entry

Clonewell

George clonewelled the digested parts. It took him FOREVER and he used a LOT of water, so he'll vacufuge the tubes tomorrow and then ligate+transform.

George's notebook entry

Transformation into BL21

Perry transformed "1ul of I13522 and I5311 samples from iGEM 2007 plates, and minipreps of T9002, S03608-I13507, S03623-I13507, J23039-T9002, I13273, diluted to ~1ng/ul, into BL21(DE3) cells."

Perry's notebook entry

7/13/07

Colony PCR

Perry colony PCR'ed the parts that he transformed into BL21:

Plate Reader ALL 96 WELLS BABY!

Stephanie set up an overnight fluorescence plate reader test with dilutions of cells instead of swabs in order to keep the number of cells more consistent between the plates. Several things were being tested on this plate. Read the entry for more information:

Stephanie's notebook entry

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