Team:BCCS-Bristol/Protocols-Testing the activity of biobrick T9002
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==Testing the activity of biobrick T9002== | ==Testing the activity of biobrick T9002== | ||
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- | + | # 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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Latest revision as of 09:57, 17 September 2008
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 (37oC, 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 10-1M (0.0021g AHL in 100μl)
- From stock solution of AHL , made up the following dilutions:
- 10-4M (1μl stock AHL in 1ml dionised H2O)
- 10-6M (1μl stock AHL in 100ml dionised H2O)
- Overnight cultures were OD'd, and diluted in LB to an OD600 of ~0.15
- In 0.5ml eppendorfs:
- 10-6M T9002 ---> 2μl 10-4M in 200μl T9002 cell suspension
- 10-6M DH5α ---> 2μl 10-4M in 200μl DH5α cell suspension
- 10-8M T9002 ---> 2μl 10-6M in 200μl T9002 cell suspension
- 10-8M DH5α ---> 2μl 10-6M in 200μl DH5α cell suspension
- Eppedorfs were vortexed gently to mix
- Tubes then incubated at 37oC, 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.