Tokyo/AHL assay
From 2007.igem.org
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===Procedure: === | ===Procedure: === | ||
| - | [[Image:AHL Assay2.JPG|thumb|200px| '''Fig.1: ''' | + | [[Image:AHL Assay2.JPG|thumb|200px| '''Fig.1: '''Externally added AHL]] |
<br>prepare overnight culture for each sample | <br>prepare overnight culture for each sample | ||
<br>make fresh culture | <br>make fresh culture | ||
| Line 28: | Line 28: | ||
<br>incubate for 2 to 3 hours | <br>incubate for 2 to 3 hours | ||
<br>apply 150 ul of samples into 96-well plaste | <br>apply 150 ul of samples into 96-well plaste | ||
| - | <br>FLA measurement | + | <br>FLA measurement |
| + | |||
===Result & Conclusion: === | ===Result & Conclusion: === | ||
<br>[[Image:hill AHL.JPG|thumb|300px| '''Fig.2: '''AHL assay graph]] | <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. | ||
Revision as of 05:21, 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:
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.
