Team:BCCS-Bristol/Protocols-Testing the activity of biobrick T9002

Testing the activity of biobrick T9002

T9002 is a luxR based receiver construct with a GFP reporter. The ability of DH5α transformed with T9002 to detect AHL and produce GFP was tested.

1. Grew up two culures (DH5α transformed with T9002 and untransformed DH5α)in LB and incubated overnight (37^oC, shaking) to log phase
2. Made stock solution of AHL: Ethyl acetate acidified with glacial acetic acid (0.01% v/v)(1μl acetic acid in 10ml ethyl acetate)AHL added to this solution to give a concentration of 10^-1M (0.0021g AHL in 100μl)
3. From stock solution of AHL , made up the following dilutions:
   1. 10^-4M (1μl stock AHL in 1ml dionised H₂O)
   2. 10^-6M (1μl stock AHL in 100ml dionised H₂O)
4. Overnight cultures were OD'd, and diluted in LB to an OD₆₀₀ of ~0.15
5. In 0.5ml eppendorfs:
   1. 10^-6M T9002 ---> 2μl 10^-4M in 200μl T9002 cell suspension
   2. 10^-6M DH5α ---> 2μl 10^-4M in 200μl DH5α cell suspension
   3. 10^-8M T9002 ---> 2μl 10^-6M in 200μl T9002 cell suspension
   4. 10^-8M DH5α ---> 2μl 10^-6M in 200μl DH5α cell suspension
6. Eppedorfs were vortexed gently to mix
7. Tubes then incubated at 37^oC, shaking for 5 minutes
8. 10μl of cell suspension was put on a microscope slide and covered with a coverslip.
9. Bright Field and GFP images were taken of each slide.