Friday 24th September
- Two experiments were set up to test the function of the BarA KO E.coli the contained the cholera system and HapR-GFP promoter in response to the cholera auto-inducer CAI-1:
1.) Simple test:
- For a simple test of the cholera system based approach the BarA KO strain which contained both the cholera system and the HapR promoter(with GFP) was grown for 1 hour at 37°C in 5ml LB (containing Cm and Amp). Then 5μL of the auto-inducer CAI-1 was added and the E.coli was left to grow again for 2 hours at 37°C.
- After the 2 hours the sample was centrifuged at 13,000rpm for 1min and excess LB was removed from the top of the sample to concentrate the sample.
- The concentrated sample was staged on a slide and looked at under a fluorescent microscope which was successfully able to identify bacteria producing GFP.
- An overnight flouresomter kinetic was set up for the co-transformed E.coli in a 96 well plate as shown bellow:
Row 1 - LB control
Row 2 - No CAI-1
Row 3 - 0.5μL CAI-1
Row 4 - 1.0μL CAI-1
Row 5 - 5μL CAI-1
Row 6 - 10μL CAI-1
Row 7 - MG1655