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

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:
 * J06702, colony #2
 * I6042, colony #1
 * I6042, colony #2
 * I13273, colony #2
 * F2622, colony #1
 * S03623 > I13507
 * S03623> E0240
 * S03608> I13507
 * F2621, colony #1
 * F2621, colony #2

Stephanie's notebook entry

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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:
 * Group 1: 1m15s extension
 * F2620
 * B0015
 * Group 2: 2m15s extension
 * I15030
 * F2620> E0240
 * F2620> I13507
 * S03068> E0240
 * Group 3: 3m15s extension
 * J37015
 * J23039 > T9002

Results: The following parts ended up being correct:
 * F2620 - colony #4
 * F2620 - colony #5
 * B0015 - colony #4
 * F2620 > E0240 - colony #2
 * J23039 > T9002 - colony #2

Stephanie's notebook entry

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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.
 * T9002, I13273, and F2620 > E0240 all either induced with 100 nM or not induced
 * J23039 > T9002
 * B0015
 * I13522
 * I5211
 * S03608 > I13507 w/ T9002
 * S03608 > I13507 w/ I13273

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:

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:
 * F2620 > E0240
 * I13273
 * J23039 > T9002
 * S03608 > I13507
 * S03623 > E0240
 * S03623 > I13507

George's notebook entry ---

Miniprepping
Stephanie miniprepped the liquid cultures that were grown on 7/11/07 in preparation for building the constitutive tetR expresser. The parts:
 * S03608 > R0040
 * S03608 > R0011
 * S03608 > P0140 colony #1
 * S03608 > P0340 colony #1
 * S03608 > R0051

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:
 * Correct
 * S03608>E0240 colony #1
 * S03608>E0240 colony #2
 * F2620>I13507 colony #1
 * F2620>I13507 colony #2
 * F2620>E0240 colony #1
 * F2620>E0240 colony #2
 * Incorrect
 * I15030 colony #1
 * I15030 colony #2
 * I15311 colony #1
 * I15311 colony #2

Stephanie's notebook entry

Sequencing Results
The results for the Genewiz sequencing from yesterday came back:
 * F2620 > E0240
 * CORRECT!
 * I13273
 * CORRECT!
 * J23039 > T9002
 * CORRECT
 * S03608 > I13507
 * CORRECT!
 * S03623 > E0240
 * CORRECT!
 * S03623 > I13507
 * CORRECT!

[[Media:071107sequences.zip]]

Digests
George performed the following digests in preparation for building the constitutive tetR generating construct:
 * XbaI and PstI digests
 * P0340
 * P0140
 * SpeI and PstI digest
 * R0011
 * R0051

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

Colony PCR
Perry colony PCR'ed the parts that he transformed into BL21:
 * J23039>T9002
 * S03623>I13507
 * T9002
 * I13522
 * S03608>B0015
 * S3608>I13507
 * I13273

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:
 * R0011 <P0140
 * R0011 <P0340
 * R0051 <P0140
 * R0051 <P0340

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
 * R0011 <P0140
 * R0011 <P0340
 * R0051 <P0140
 * R0051 <P0340

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

George's notebook entry

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:
 * R0011
 * R0051
 * R0052

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

Stephanie's notebook entry

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
 * R0011+P0140
 * R0051+P0140
 * R0052+P0140
 * R0011+P0340
 * R0051+P0340
 * R0052+P0340

George's notebook entry

Transformation of the R-P constructs
George transformed the into Top10 and plated on LB carb plates.
 * R0011+P0140
 * R0051+P0140
 * R0052+P0140
 * R0011+P0340
 * R0051+P0340
 * R0052+P0340

George's notebook entry