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 (37oC, 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:
 * 4) 10-4M (1μl stock AHL in 1ml dionised H2O)
 * 5) 10-6M (1μl stock AHL in 100ml dionised H2O)
 * 6) Overnight cultures were OD'd, and diluted in LB to an OD600 of ~0.15
 * 7) In 0.5ml eppendorfs:
 * 8) 10-6M T9002 ---> 2μl 10-4M in 200μl T9002 cell suspension
 * 9) 10-6M DH5α ---> 2μl 10-4M in 200μl DH5α cell suspension
 * 10) 10-8M T9002 ---> 2μl 10-6M in 200μl T9002 cell suspension
 * 11) 10-8M DH5α --->  2μl 10-6M in 200μl DH5α cell suspension
 * 12) Eppedorfs were vortexed gently to mix
 * 13) Tubes then incubated at 37oC, shaking for 5 minutes
 * 14) 10μl of cell suspension was put on a microscope slide and covered with a coverslip.
 * 15) Bright Field and GFP images were taken of each slide.