Tokyo/AHL assay

From 2007.igem.org

(Difference between revisions)
(Result & Conclusion:)
(Procedure:)
Line 24: Line 24:
<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) = 10 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>[IPTG]final (in 3 ml LB culture) = 1 mM  
<br>[IPTG]final (in 3 ml LB culture) = 1 mM  
<br>incubate for 2 to 3 hours  
<br>incubate for 2 to 3 hours  

Revision as of 05:42, 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
check how lacI represses 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:

Fig.1: Externally added AHL


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:


Fig.2: Result of AHL assay


AND gate by AHL & IPTG
Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG.