IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 3

Sequencing Parts
Stephanie Nanodropped and sent the following parts for sequencing:
 * S03623
 * S03608
 * J37034
 * J23039
 * R0011
 * R0140
 * T9002
 * B0015
 * R0051

Stephanie's notebook entry

Constructing Growth Curves
Stephanie used the plate reader and Bl21+I13263 cells diluted 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, and 1:100 as well as to construct a growth curve of OD vs time.

Stephanie's notebook entry

Liquid Cultures
Stephanie grew liquid cultures of I5311, OmpA1+his, I13522, T9002, and I13263 in preparation for induction on 07/03/07.

Stephanie's notebook entry

Digestion
Perry performed the following digests:
 * R0011 with SpeI and PstI
 * R0040 with SpeI and PstI
 * R0051 with SpeI and PstI
 * S03608 with EcoRI and SpeI
 * S03623 EcoRI and SpeI
 * J37034 XbaI and PstI (x2)
 * B0015 EcoRI and SpeI

Perry and Stephanie also ran a gel and gel-extracted the result. However, the J37034 did not turn out as expected so Stephanie ran another digest on the J37034

Perry's notebook entry

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Results of the Growth Curves
Results posted by Stephanie

Digests
Perry and Stephanie digested the E0240, S03608, S03623, and B0015 parts in order to rebuild the J37034.

Stephanie's notebook entry Perry's notebook entry

Ligation and Transformation of Parts
Perry and Stephanie ligated with Quick Ligase: 7ul S03608/S03623 + 3ul B0015 in pSB1AK3. They then transformed the ligated parts into 20ul Top10.

Perry's notebook entry

Plate Reader Fluorescence Setup
Stephanie set up a plate reader experiment for fluorescence.

"First, I loaded the machine with cells (dilution - 1:10 ... maybe will dilute in greater amounts next time, eg 1:100?) and watched OD. Around 0.3OD I took them out, transferred to a clear/black 96-well plate (next time, will start with one of these), and induced cells with OHHL."

Stephanie's notebook entry

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07/04/07
Stephanie transferred plates of the ligations from the incubator to the fridge. She also checked the results from the plate reader fluorescent readings.

Stephanie's notebook entry

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Colony PCR
Stephanie prepared a colony PCR of several colonies of S03608-B0015 and S03623-B0015

Stephanie's notebook entry

The results were not good.

Results posted by Perry (the first gel)

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Colony PCR of F2620
Stephanie and Perry prepared and ran a colony PCR of F2620

Perry's notebook entry (including results - second gel)

Digestion, Ligations, and Transformations, Oh my!
Stephanie and Perry digested and ligated the following parts: where X>Y indicates that the piece X was ligated into the larger piece Y, containing the vector.
 * F2620>I13507
 * S03608>E0240
 * S03623>I13507
 * S03623>E0240
 * F2620>E0240
 * S03608>I13507

Stephanie and Perry also transformed all the above parts into Top10 cells. In addition, they transformed the following parts into Top10 cells:
 * F2621
 * I15030
 * J37015
 * J06702
 * I6042
 * I13273
 * F2622