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

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==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.


# Grew up two culures (DH5α transformed with T9002 and untransformed DH5α)in LB and incubated overnight (37<sup>o</sup>C, shaking) to log phase
# 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<sup>-1</sup>M (0.0021g AHL in 100μl) 
# From stock solution of AHL , made up the following dilutions:
##10<sup>-4</sup>M (1μl stock AHL in 1ml dionised H<sub>2</sub>O)
##10<sup>-6</sup>M (1μl stock AHL in 100ml dionised H<sub>2</sub>O)
# Overnight cultures were OD'd, and diluted in LB to an OD<sub>600</sub> of ~0.15
# In 0.5ml eppendorfs:
##10<sup>-6</sup>M T9002 ---> 2μl 10<sup>-4</sup>M in 200μl T9002 cell suspension
##10<sup>-6</sup>M DH5α  ---> 2μl 10<sup>-4</sup>M in 200μl DH5α cell suspension
##10<sup>-8</sup>M T9002 ---> 2μl 10<sup>-6</sup>M in 200μl T9002 cell suspension
##10<sup>-8</sup>M DH5α  --->  2μl 10<sup>-6</sup>M in 200μl DH5α cell suspension
# Eppedorfs were vortexed gently to mix
# Tubes then incubated at 37<sup>o</sup>C, shaking for 5 minutes
# 10μl of cell suspension was put on a microscope slide and covered with a coverslip.
# Bright Field and GFP images were taken of each slide.

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