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Contents 1 Lab Session 12/08/09 1.1 Stochastic Switch Team 1.2 Sporulation Tuning/Chassis Team 1.2.1 Protocol

Lab Session 12/08/09

Stochastic Switch Team

Today we are trying to transform Bacillus subtilis with gfp-rrnb integration vector. Metal sensor team kindly inoculated B. subtilis cells into flask tubes and placed them into the shaking incubator. We labelled them as 1,2,3 and control.

We prepared four plates

• LB + Bsubtilis (+ Control, cells should grow on this plate)
• LB + CHL + B. subtilis (- control, cells will not grow on this plate)
• LB + CHL + B. subtilis + plasmid DNA(Cells should transform with the DNA)
• LB + CHL + B. subtilis + diluted plasmid DNA (Cells should transform with the DNA)

• 2xcompetence medium and 2xstravation medium were prepared.
• We used the 2nd and the 3rd tubes for the tests
• Incubated the samplesfor three hours at 37C
• Added the starvation medium and incubated for another 2 hours
• For each of the two cultures, added 0.4ml of the final solution from the culture and 10ul of DNA into an eppendorf tube.
• Placed the tubes in the shaking incubator for an hour. To do this we taped the tubes to the base of the incubator. N.B- Chris in the lab swapped the temperatures of the incubators, so we used the one that is opposite the -80 freezer. The starter overnight cultures are also in this incubator -these are labelled 1,2,3 control (we used 2 and 3).
• The cultures were plated out using glass beads and put in the 37 incubator.

Sporulation Tuning/Chassis Team

Today, we made our first attempt to recover the cwlD spores which were kindly sent to us by Anne Moir from Sheffield University as mentioned on the 4th of August.

We used the protocol for Method A as mentioned.

Protocol

• 10ul of cwlD spores were added into the lysozyme and buffer solution.