Tokyo/AHL assay

__NOTOC__
<br>[[Tokyo/Works|Works top]]&nbsp;&nbsp;&nbsp;0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]]&nbsp;&nbsp;&nbsp;1.[[Tokyo/Works/Formulation |Formulation]]&nbsp;&nbsp;&nbsp;'''2.[[Tokyo/Works/Assay |Assay1]]'''&nbsp;&nbsp;&nbsp;3.[[Tokyo/Works/Simulation |Simulation]]&nbsp;&nbsp;&nbsp;4.[[Tokyo/Works/Assay2 |Assay2]]&nbsp;&nbsp;&nbsp;5.[[Tokyo/Works/Future works |Future works]]
<br><br>
[[Tokyo/Hill function fitting |Objective of this assay]]&nbsp;&nbsp;&nbsp;[[Tokyo/AHL assay|Effect of AHL]]&nbsp;&nbsp;&nbsp;[[Tokyo/IPTG assay|Effect of IPTG]]　　[[Tokyo/Preliminary assays |Preliminary assays]]

== Effect of AHL ==

===Objective: ===
<br>By testing how AHL activates lux-lac hybrid promoter, parameters(n2,K2) should be determined. 

<!--<br>check how lacI represses lux-lac hybrid promoter-->

===Samples: === in DH5 alpha
<br>placIQ(constitutive promoter) + GFP (Pos. con.) :[http://partsregistry.org/Part:BBa_J54201 BBa_J54201]
<br>No promoter + GFP (Neg. con.) :[http://partsregistry.org/Part:BBa_J54102 BBa_J54102]
<br>Lux lac hybrid promoter + GFP :[http://partsregistry.org/Part:BBa_I751900 BBa_I751900]

<!--<br>hybrid promoter plasmid with pTrc99A 
<br>hybrod promoter plasmid with pBR322 
<br>luxR plasmid with pTrc99A 
<br>luxR --- AHL-dependent activation confirmed 
<br>placIq on promter-less GFP in DH5a (for pos. con.) 
<br>promoter-less GFP in DH5a (for neg. con.)-->

===Procedure: ===
[[Image:AHL Assay2.JPG|thumb|200px| '''Fig.1: '''Activated by externally added AHL, cells had accumulated GFP, increasing fluorescence intensity.]]
<br>prepare overnight culture for each sample
<br>make fresh culture 
<br>take 3 ul of the overinight culture into 3 ml of LB (+ Amp and Kan) in Falcon tubes. 
<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>The final concentration of AHL (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM 
<br>(*The same amount of DMSO, solvent for AHL, was added to each sample.)
<br>incubate for 2 to 3 hours 
<br>apply 150 ul of samples into 96-well plaste 
<br>FLA measurement

===Result & Conclusion: ===
[[Image:hill AHL.JPG|thumb|300px| '''Fig.2: Result of AHL assay'''<br>Fluorescence intensity (arbitrary unit, a.u.) as a function of the concentration of AHL was determined. ]]
<br>The concentration of AHL dose-denpendently increased iGFP fluorescence. This result indicates that the hybrid promoter’s activation is strengthend with increasing concentration of AHL. From the activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), is determined.

<br>'''n2 = 2.08 (-)'''
<br>'''K2 = 4.05 (μM)'''


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