From 2009.igem.org

From left to right, top row: 1mM IPTG with 0.075 mg/mL tyrosine, 1mM IPTG and 0.3 mg/mL tyrosine, and then 1mM IPTG and 0.6 mg/mL tyrosine From left to right, bottom row: 0.075 mg/mL tyrosine, 0.3 mg/mL tyrosine, and then 0.6 mg/mL tyrosine.

It appears that the bacteria were much too high a concentration; they formed lawns. As we saw on the first plates, pigment production is reduced at high bacterial concentrations. However, it is encouraging that the darkest plate is the one with the greatest tyrosine concentration and IPTG.

To follow up, the experiment was repeated with 1/100 and 1/1000 dilutions of bacterial culture on the following plates:

100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine

100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine

100ug/ml Ampicillin, 7.5 ug/ml CuSO4, 0.075 mg/mL tyrosine, 0.5mM IPTG

100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG

100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine, 1mM IPTG

Unfortunately, the overnight cultures for the plate readers were contaminated, so a single colony was once again incubated overnight in 10 mL of 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG in preparation for pigment characterisation using the plate reader.

Amplification

Chose 5 activator / promoter combinations to continue work with:

P2 ogr activator with

PO promoter (I1746371)
Psid promoter (I746374)

phiR73 delta activator with

PF promoter (I746390)
PO promoter (I746391)
Psid promoter (I74394)

Of the above, on plates all showed leaky expression of GFP and RFP, suggesting that last weeks transformation was successful.

Inoculated 5 replicates of single colonies overnight of each in preparation for colony PCR and miniprep tomorrow.

Team:Cambridge/Notebook/Week4

From 2009.igem.org

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Week 4 - Development

Monday

Wet Work

Melanin

Amplification

Dry Work