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Lab Session 13/08/09

Sporulation Tuning/Chassis Team

Yesterday's germination attempt seems to have worked, but results are not yet clear. This may have been due to a calculation error for the lysozyme.

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 samples for three hours at 37C
• Added the starvation medium and incubated for another 1 hour 40 minutes
• 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 45 mins. To do this we taped the tubes to the base of the incubator.
• The cultures were plated out using glass beads and put in the 37 incubator.