Team:PKU Beijing/Notebook/Protocol/Chemical Inducible GFP

Protocol for chemical inducible expression of GFP

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Materials:

• 4 groups of induce solution with a concentration gradient of <math>10^{-7}, 10^{-5}, 10^{-3}, 10^{-2}</math>;
• Overnight bacterial culture or bacterial colonies;
• Phosphate Buffered Solution (PBS).

Procedure:
1. Add 20 μl of the overnight bacterial culture or pick a colony to 5ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.
2. Add 0.5 mL of the fresh bacterial culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of <math>10^{-9}</math>, <math>10^{-8}, 10^{-7}, 10^{-6}, 10^{-5}, 10^{-4}, 10^{-3}, 10^{-2}</math>.
3. Place the induction system at 37 degree for 2 hours.
4. Pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant.
5. Resuspend the pelleted cells in 500 μl of PBS.
6. Transfer 100 uL of bacterial resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.

Note:
If desired, time sequential expression of GFP can also be tested, through verifying the incubating time of induction system at 37 degree.

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