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Wet lab work was carried out from 4 August to 19 September, Mondays to Fridays. Please click on a day to see the lab notebook.

Friday 12th September

The ON cultures of iGEMgfp and Bs168 were diluted by adding 250μl ON culture to 10mL LB and incubating at 37°C whilst shaking for 2 hours. The two diluted cultures were then each split into 5 x 2mL aliquots and subtilin added in the following concentrations:

- 0% induction (2mL dilute culture)

- 0.1% induction (2mL dilute culture + 2μl subtilin)

- 0.5% induction (2Ml dilute culture + 10μl subtilin)

- 1% induction (2mL dilute culture + 20μl subtilin)

-10% induction (2mL dilute culture + 200μl subtilin)

The five Bs168 tubes acted as controls. These were incubated at 37°C whilst shaking for 1 hour to allow the subtilin to induce gfp expression.

Plasmid was isolated from the 12 pGFPrrnB-BB107 ON cultures 9see protocol). Unfortunately plasmid from colony 4 (25μl plate) and colony 4 (250μl plate) were lost due to human error.

pGFPrrnB-ncl08 colony 7 and pJWV021 colony 13 isolated from overnight cultures (see protocol).

All isolated plasmid was restricted using 7.5μl plasmid and 0.5μl each enzyme in 15μl total volume:

- pGFPrrnB-BB107 restrited with EcoRI and SpeI.

- pGFPrrnB-ncl08 restricted with EcoRI and NheI

- pJWV021-ncl08 restricted with NheI and BglII

These restrictions were run on gels.

Lane 1: 1kb ladder

Lane 2: pGFPrrnB-BB107 colony 1

Lane 3: pGFPrrnB-BB107 colony 2

Lane 4: pGFPrrnB-BB107 colony 3

Lane 5: pGFPrrnB-BB107 colony 5

Lane 6: pGFPrrnB-BB107 colony 6

Lane 7: pGFPrrnB-BB107 colony 7

Lane 8: pGFPrrnB-BB107 colony 8

Lane 9: pGFPrrnB-BB107 colony 9

Lane 10: pGFPrrnB-BB107 colony 10

Lane 11: pGFPrrnB-BB107 colony 11

Lane 1: 1kb ladder

Lane 2: pGFPrrnB-ncl08 colony 7

Lane 3: pJWV021-ncl08 colony 13

Lane 4: pGFPrrnB-ncl08 colony 7 control (no enzyme)

Lane 5: pJWV021-ncl08 colony 13 control (no enzyme)