Newcastle University Wetlab/11 September 2008

Thurday 11th September

 * Single colonies of iGEMcherry were streaked from the overnight kanamycin plates onto sucrose and glucose plates.


 * Stab cultures were taken from the overnight 25μl and 250μl pGFP-BBa_I7045898 plates (grown on spectinomycin) and grown in 10mL LB:

- 25μl plate white colony 1

- 25μl plate white colony 2

- 25μl plate white colony 3

- 25μl plate white colony 4

- 25μl plate white colony 5

- 25μl plate white colony 6

- 250μl plate white colony 1

- 250μl plate white colony 2

- 250μl plate white colony 3

- 250μl plate white colony 4

- 250μl plate white colony 5

- 250μl plate green colony 6

These were incubated overnight at 37˚C whilst shaking.


 * iGEMgfp was diluted by adding 250μl of the overnight culture to 10mL LB in a sterile shake flask. This was incubated at 37˚C whilst shaking for a further 2 hours to give the cells fresh nutrients and allow them to multiply. This culture was then divided into 3 tubes (3mL in each) and subtilin added in the following concentrations:

- 0% induction (3mL dilute iGEMgfp)

- 1% induction (3mL dilute iGEMgfp + 30μl subtilin)

- 10% induction (30mL dilute iGEMgfp + 300μl subtilin)

These were incubated whilst shaking for 1 hour at 37°C. Flow cytometry was then performed on the samples.


 * Overnight cultures were made from glycerol stocks pGFPrrnB-ncl08 colony 7 and pJWV021-ncl08 colony 13 (see protocol). The plasmid from these cultures are to be checked for the 2.2kb fragment before being sent for sequencing.