IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 4

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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:

This was done in preparation for making the constitutive tetR+JT construct.

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:

This was done in preparation for making the constitutive tetR+JT 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.

This was done in preparation for making the constitutive tetR+JT construct.

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

Dephosphorylation of P0140, P0340, R0011, and R0051

George vacufuged the results of the clonewell on 7/12/07 down to a high concentration. Then he treated the DNA with Antarctic Phophatase. Finally, he heat inactivated the mix (10 min at 65dC).

This was done in preparation for making the constitutive tetR+JT construct.

George's notebook entry

Ligation of P0140, P0340, R0011, and R0051

George Roche ligated:

This was done in preparation for making the constitutive tetR+JT construct.

George's Notebook Entry

Transformation of constitutive tetR constructs

George transformed

into BL21

This was done in preparation for making the constitutive tetR+JT construct.

George's notebook entry

7/14/07

Colony PCR of R-P constructs

Stephanie colony PCR'ed the ligated constructs that George transformed into BL21. The results were very bad, as in there were no bands.

This was done in preparation for making the constitutive tetR+JT construct, but it didn't work. So they'll redo everything starting tomorrow.

Perry's notebook entry

Stephanie's notebook entry

Liquid cultures of R0011, R0051, and R0052

Stephanie grew liquid cultures of the three promoter parts:

Basically, it is redoing the same sequence of digestion, ligation, etc. except we have added in another promoter, R0052.

Stephanie's notebook entry

7/15/07

Miniprepping R0011, R0051, and R0052

Stephanie miniprepped the overnight liquid cultures in preparation for rebuilding the PR+JT part. Stephanie's notebook entry

Digesting the R0011, R0051, and R0052

Stephanie digested 38 µL each of R0011, R0051, and R0052 with SpeI and PstI.

Stephanie's notebook entry

Dephosphorylating the R0011, R0051, and R0052

George dephosphorylated R0011, R0051, and R0052 with Antarctic Phosphotase.

George's notebook entry

Clonewell of R0011, R0051, and R0052

George clonewelled (blech...) the R0011, R0051, and R0052.

George's notebook entry

Vacufuge of R0011, R0051, R0052

George vacufuged his sample from the clonewell.

George's notebook entry

Ligation or P0140, P0340, R0011, R0051, R0052

George ligated

George's notebook entry

Transformation of the R-P constructs

George transformed the

into Top10 and plated on LB carb plates.

George's notebook entry

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