Tokyo/Repression and activation check with LacI and AHL
From 2007.igem.org
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<br>FLA measurement | <br>FLA measurement | ||
===Result & Conclusion: === | ===Result & Conclusion: === | ||
- | <br>[[Image:hill AHL.JPG|thumb| | + | <br>[[Image:hill AHL.JPG|thumb|300px| '''Fig.2: '''AHL assay graph]] |
<br>AND gate by AHL & IPTG | <br>AND gate by AHL & IPTG | ||
<br>Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG. | <br>Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG. |
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:
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.