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Team:Cambridge/Notebook/Week4
From 2009.igem.org
Categories :
Project :
-
Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
Week 4
Monday
Wet Work
Melanin
- Results from weekend plates:
<need a picture>
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.
Ampiflication
- 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.
