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_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 | *[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 | ||
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] | *[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] | ||
[[User:ShLo#7.2F12|Stephanie's notebook entry]] | |||
====Colony PCR==== | |||
Stephanie and George colony PCR'ed some parts: | |||
*Correct | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]>[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240] colony #1 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]>[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240] colony #2 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620]>[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507] colony #1 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620]>[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507] colony #2 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620]>[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240] colony #1 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620]>[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240] colony #2 | |||
*Incorrect | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_I15030 I15030] colony #1 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_I15030 I15030] colony #2 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_I15311 I15311] colony #1 | |||
**[http://parts.mit.edu/registry/index.php/Part:BBa_I15311 I15311] colony #2 | |||
[[User:ShLo#7.2F12|Stephanie's notebook entry]] | [[User:ShLo#7.2F12|Stephanie's notebook entry]] |
Revision as of 21:06, 12 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:
- 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
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}),
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
- Group 2: 2m15s extension
- Group 3: 3m15s extension
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
07/11/07
Setting Up the Overnight Fluorescent Plate Reader
Stephanie set up the plate reader for an overnight plate reader experiment with:
- 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
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.
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."
FACS result
7/12/04
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!
Miniprepping
Stephanie miniprepped the liquid cultures that were grown on 7/11/07 in preparation for building the constitutive tetR expresser. The parts:
Colony PCR
Stephanie and George colony PCR'ed some parts:
- Correct
- Incorrect