Tokyo/AHL assay
From 2007.igem.org
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===Purpose: === | ===Purpose: === | ||
<br>check how AHL activates lux-lac hybrid promoter | <br>check how AHL activates lux-lac hybrid promoter | ||
| - | <br>check how lacI represses lux-lac hybrid promoter | + | <!--<br>check how lacI represses lux-lac hybrid promoter--> |
===Samples: === | ===Samples: === | ||
Revision as of 05:58, 25 October 2007
Abstract Concept & Model Requirements Genetic_circuit Works About_our_team
Works top 0.Hybrid promoter 1.Formulation 2.Assay1 3.Simulation 4.Assay2 5.Future works
AHL assay
Purpose:
check how AHL activates lux-lac hybrid promoter
Samples:
hybrid promoter plasmid with pTrc99A
hybrod promoter plasmid with pBR322
luxR plasmid with pTrc99A
luxR --- AHL-dependent activation confirmed
placIq on promter-less GFP in DH5a (for pos. con.)
promoter-less GFP in DH5a (for neg. con.)
Procedure:
prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (Amp and/or 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
[IPTG]final (in 3 ml LB culture) = 1 mM
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.
