Tokyo/Repression and activation check with LacI and AHL

From 2007.igem.org

Latest revision as of 05:03, 24 October 2007

AHL assay

Purpose:

check if AHL activates lux-lac hybrid promoter
check if lacI represses lux-lac hybrid promoter

Samples:

hybrid promoter in pTrc99A
hybrod promoter in pBR322
luxR in pTrc99A
luxR --- AHL-dependent activation confirmed
placqi on promter-less GFP in DH5a (for pos. con.)
promoter-less GFP in DH5a (for neg. con.)

Procedure:

Fig.1: Exogenously 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) = 10 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: AHL assay graph

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