Team:PKU Beijing/Parts Characterization/BBa K228009/protocol

BBa K228009 - AraC protein(reversed sequence) and Pbad promoter

Designed by Rengcheng Gao Group: iGEM09_PKU_Beijing (2009-09-18)
Input: Arabinose molecules
Output: GFP fluorescence

Protocol

Materials
A subset of inducer solution with a concentration gradient of 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3;
Bacterial colonies;
Phosphate Buffered Solution (PBS).
Procedure
1. One 40 ml culture of LB medium and antibiotic (Ampicillin, 100ng/ml) was inoculated with a single colony from a LB agar plate containing the report system mentioned above.
2. Cultures were grown in 1.5 ml test tubes for several hrs at 37°C with shaking at 70 rpm to an OD600 of 0.40. This growth took 4 hrs on average.
3. Add 1.5 mL of the fresh bacteria culture to 42 new test tubes. Then pipet appropriate volume of arabinose solution into test tubes to yield 7 different final concentrations (0 control, 1.0*10^-8, 1.0*10^-7, 1.0*10^-6, 1.0*10^-5, 1.0*10^-4, 1.0*10^-3). Thus 6 replicates were measured for each concentration of arabinose. The concentration of 0 control group is to measure fluorescent background.
4. Place the induction system at 37 degree. Every 20 minites, pipet 200 uL of the culture from each 1.5 ml test tube into a new 1.5 ml test tube, respectively, till the incubating time reaches 2 hrs.
5. Pellet bacteria cells by 1min centrifugation at 13000 rpm, and discard the supernatant. Resuspend the pelleted cells in 200ul of PBS. Time between repeated operations was about 20mins.
6. Transfer 100 uL of bacterial resuspension into each well of 96-well plate to test the GFP fluorescence by Microplate Reader.
7. Use spectrophotometer to test the OD600 value of the left 100 ul of bacterial resuspension. 8. We converted the OD600 value into the concentration of cells (/ml). The equation is OD600 1.0=5*10^8 cells/ml. Then we normalized the GFP fluorescence by the OD600 value, to obtain the ratio of GFP fluorescence to the OD600 value. The mean for each concentration-time group was then averaged across 6 replicates to obtain a population mean.
9. The transfer function in Figure 1 is the 120 min time-slice from the time and dose dependent input-output surface. The data points represent the mean of 6 individual measurements. The corresponding error bars denote the 95% confidence interval in the mean of the independent measurements.
10. To estimate parameters that characterize the measured transfer functions, we used least squares estimation to fit a simple model to the data. Hill equations derived from simple biochemical equations describe the data well (Table 1). Pmax is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.

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