Tokyo/Repression and activation check with LacI and AHL
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<br>promoter-less GFP in DH5a (for neg. con.) | <br>promoter-less GFP in DH5a (for neg. con.) | ||
===Procedure: === | ===Procedure: === | ||
+ | [[Image:AHL Assay2.JPG|thumb|200px| '''Fig.1: '''Exogenously added AHL]] | ||
<br>prepare overnight culture for each sample | <br>prepare overnight culture for each sample | ||
<br>make fresh culture | <br>make fresh culture |
Revision as of 04:59, 24 October 2007
Contents |
Repression and activation check with LacI and AHL
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:
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:
AND gate by AHL & IPTG
Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG.