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Contents 1 Lab Session 10/08/09 1.1 Stochastic Switch Team 1.1.1 Results 1.1.1.1 Sample 1 1.1.1.2 Sample 2 1.2 Chassis team 1.3 Metal Sensor Team 1.3.1 Summary 1.3.2 Protocol 1.3.3 Results

Lab Session 10/08/09

Stochastic Switch Team

Today we pepared gfp-rrnb plasmids using midiprep. We followed GenElute's midiprep protocol.

We had two E.coli cultures inoculated with gfp-rrnb left overnight. Although the samples were of the same plasmid, we treated them individually as one culture seemed to express the GFP more than the other. We used 100ml of each sample aliquoted into two 50ml falcon tubes then carried out the midi prep procedure from the protocol on the 4 tubes, combining our DNA samples at the end. In the DNA concentration step we centrifuged the tubes at 16000rpm for 30 minutes before adding ethanol to rinse the DNAs. We then centriguged the tubes again for 10 minutes at 16000rpm.

We evaporated the tubes for 5 minutes at the end before resuspending each tube in 50ul of distilled water. The tubes from the same cultures were then combined and the DNA concentrations for the two samples were measured using the spectrometer.

Results

We got good DNA concentrations for both samples. The results are below:

Sample 1

The concentration of DNA: 829.6 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.88
The ratio of protein concentration to RNA concentration(260/230): 2.33

Sample 2

The concentration of DNA: 601.1 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.90
The ratio of protein concentration to RNA concentration(260/230): 2.11

Chassis team

• Created germination solution without lysozyme.
• Borrowed Em from Colin Harwood's lab.
• Created a new MgCl Stock solution, as old one had bits of matter floating in it.
• Created KPh stock solution.
• Autoclaved solutions.
• Made 250ml LB and 250ml H20, autoclaved.
• Inoculated LB with Em and Kan, then poured plates.

Metal Sensor Team

Summary

Preparation for test transformation in Bacillus subtilis 168. This afternoon we prepared 40 ml MM competence media and equally distributed it into four 15ml falcon tubes. To three of the tubes (one tube for each of the three teams), Bacillus subtilis 168 was added and to the fourth tube, nothing was added - this would serve as contamination control. They were then left in the shaking incubator at 37ºC overnight for the transformation process tomorrow.

Protocol

For the preparation of the 40ml MM competence media (10ml in each of 4 falcon tubes), please refer to the solutions list. For the steps regarding preparations of overnight cultures, please refer to the 'Evening before transformations day' section. The only changes to the protocol were: 4 falcon tubes were used instead of just 2 - three tubes contained Bacillus subtilis innoculated MM competence media (for the three sub-teams in our project) and the fourth tube contained MM competence media alone (contamination control)

Results

3 x 15ml falcon tubes containing 10ml MM competence media along with Bacillus subtilis cells and 1 x 15ml falcon tube containing just 10ml MM competence media. All four tubes placed in shaking incubator for overnight growth.