IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 2

Transformation of Parts into Top10
See Perry's Transformation Protocol, transforming the parts F1610, I13263, and I13272 into Top10 cells

Plating of Top10 cells containing the parts
We plated the three parts onto three separate petri dishes.

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Growth of colonies in liquid cultures
We grew a large amount because we want to carry out a midiprep tomorrow
 * 1) Take 3 500 mL flasks from the cabinet in the big room with the house on the chalkboard and the person saying "Yaye"
 * 2) Put 150 mL of LB and 150 µL of ampicillin (all the parts are ampicillin resistant) into each flask
 * 3) Put into the agitating incubator overnight

Midiprep with the liquid cultures grown on 6/26
See HiSpeed Midi and Maxi Prep for protocol used

Transformation of I13263 into BL21
See Transformation Protocol for the protocol used.

Sequencing the I13263
See Sequencing Protocol for the protocol used.

Details of the order:

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F1610 Checks

 * F1610 Digestion Part I
 * George's Noteboook Entry
 * Perry and George digested the F1610 with XbaI and PstI.
 * Clonewell
 * F1610 is about 800 bp, there were no visible bands in that region. There is something wrong.
 * F1610 Digestion Part II (more DNA)
 * George's Notebook Entry
 * Stephanie ran the same digestion on the F1610 except with more DNA.

Growing Liquid Cultures of I13263
Stephanie and George grew liquid cultures of I13263 in preparation for the FACS appointment.

Sequences of F1610, I13263, I13272
George's Noteboook Entry

Perry checked the Genewiz sequencing results for the F1610, I13263, and I13272. The results for F1610 were not good, excellent for I13263, and for I13273.

Transformation of Additional Parts
Perry and George transformed the following parts
 * R0040
 * Promoter
 * R0051
 * Promoter
 * R0011
 * Promoter
 * E0240
 * RBS+GFP+Terminator
 * B0015
 * Terminator
 * T9002
 * Complete luxR+GFP system

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FACS appointment
We diluted and grew the overnight samples to log phase (in a very sketchy manner) and induced the samples with 1, 10, and 100 nM HSL 1, 2, and 4 hours before the FACS appointment.

The results were disappointing. We didn't have any detectable fluorescence in any of the samples.

George's notebook entry Stephanie's notebook entry

Even more BioBricks
Perry and George transformed and grew the following parts in liquid culture:
 * S03623
 * S03608
 * J37034
 * J23039
 * R0011
 * R0140
 * T9002
 * B0015
 * R0051

George's notebook entry ---

Miniprepping R0011, B0015, T9002
Stephanie miniprepped all the parts mentioned in the previous section. Unfortunately, the labels for R0011, B0015, and T9002 smeared so she ran an E-gel to distinguish between the three.

Stephanie's notebook entry ---

E-gel results
Stephanie and Perry looked at the results of the E-gel to distinguish between the three samples.

Stephanie's notebook entry