Team:PKU Beijing/Parts Characterization/BBa K228258/Protocol

Designed by Guosheng Zhang Group: iGEM09_PKU_Beijing
Input: Arabinosem and salicylate molecules
Output: GFP fluorescence

Protocol for Fluorescent Test

• Materials

7 groups of inducer solution with a concentration gradient of 10^-1M, 10^-2 M, 10^-3 M, 10^-4 M, 10^-5 M, 10^-6 M and control (no inducer) for both arabinose and salicylate;

Bacterial colonies;

Water.

• Procedure

1. 30 mL culture of LB medium with antibiotic (A+K+) was inoculated with a single colony from a LB agar plate containing the AND gate plasmid BBa_K228258 (the constructed report system described in the above).
2. Cultures were grown in a conical breaker for several hours at 37°C with shaking at 70 rpm until the OD600 value is around 0.400. This growth took 4 hours on average.
3. Add 0.5 mL of the fresh bacteria culture respectively to 49 sterilized EP tubes. Then pipet 5uL corresponding arabinose and salicylate solution into EP tubes to yield 7 different final concentrations for both arabinose and salicylate(0, 1.0*10^-8M, 1.0*10^-7M, 1.0*10^-6M, 1.0*10^-5M, 1.0*10^-4M, 1.0*10^-3M). The control (concentration=0) group is set to measure the fluorescent background.
4. Place the induction system at 37 degree for 2 hours.
5. Pellet bacteria cells by 4min centrifugation at 3000 rpm, and discard the supernatant as possible as we could. Resuspend the pelleted cells with 500ul of water.
Transfer 100uL of bacteria resuspension into each well of 96-well plate to test the GFP fluorescence by Microplate Reader.
7. Pipet 100 uL of the bacteria resuspension from each 1.5 mL EP tube and use the spectrophotometer to test the OD600 value.
8. Convert the OD600 value into the concentration of cells (/mL). The corresponding relation is OD600 1.0=5*10^8 cells/ml. Then we normalized the GFP fluorescence by calculating the ratio of absolute GFP fluorescence to the OD600 value.

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