Tokyo/AHL assay
From 2007.igem.org
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<br>prepare overnight culture for each sample | <br>prepare overnight culture for each sample | ||
<br>make fresh culture | <br>make fresh culture | ||
| - | <br>take 3 ul of the overinight culture into 3 ml of LB (Amp and | + | <br>take 3 ul of the overinight culture into 3 ml of LB (+ Amp and Kan) in Falcon tubes. |
<br>incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter) | <br>incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter) | ||
<br>add AHL & IPTG solution | <br>add AHL & IPTG solution | ||
<br>[AHL]final (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM | <br>[AHL]final (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM | ||
| - | + | ||
<br>incubate for 2 to 3 hours | <br>incubate for 2 to 3 hours | ||
<br>apply 150 ul of samples into 96-well plaste | <br>apply 150 ul of samples into 96-well plaste | ||
Revision as of 10:59, 25 October 2007
Works top 0.Hybrid promoter 1.Formulation 2.Assay1 3.Simulation 4.Assay2 5.Future works
Purpose of this assay 1.AHL assay 2.IPTG assay Preliminary assays
AHL assay
Purpose:
To check how AHL activates the newly devised lux-lac hybrid promoter
Samples:
Procedure:
prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (+ Amp and Kan) in Falcon tubes.
incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter)
add AHL & IPTG solution
[AHL]final (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement
Result & Conclusion:
As the concentration of AHL increases, GFP fluorescence increased, indicating that the hybrid promoter’s activation is strengthend with increasing concentration of AHL. From the activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), is determined.
