Tokyo/AHL assay
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| - | <br>[[Tokyo/Works|Works top]] | + | <br>[[Tokyo/Works|Works top]] 0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]] 1.[[Tokyo/Works/Formulation |Formulation]] '''2.[[Tokyo/Works/Assay |Assay1]]''' 3.[[Tokyo/Works/Simulation |Simulation]] 4.[[Tokyo/Works/Assay2 |Assay2]] 5.[[Tokyo/Works/Future works |Future works]] |
<br><br> | <br><br> | ||
| - | [[Tokyo/Hill function fitting |Objective of this assay]] | + | [[Tokyo/Hill function fitting |Objective of this assay]] [[Tokyo/AHL assay|Effect of AHL]] [[Tokyo/IPTG assay|Effect of IPTG]] [[Tokyo/Preliminary assays |Preliminary assays]] |
== Effect of AHL == | == Effect of AHL == | ||
===Objective: === | ===Objective: === | ||
| - | <br> | + | <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--> | <!--<br>check how lacI represses lux-lac hybrid promoter--> | ||
| - | ===Samples: === | + | ===Samples: === in DH5 alpha |
<br>placIQ(constitutive promoter) + GFP (Pos. con.) :[http://partsregistry.org/Part:BBa_J54201 BBa_J54201] | <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>No promoter + GFP (Neg. con.) :[http://partsregistry.org/Part:BBa_J54102 BBa_J54102] | ||
Latest revision as of 02:45, 27 October 2007
Works top 0.Hybrid promoter 1.Formulation 2.Assay1 3.Simulation 4.Assay2 5.Future works
Objective of this assay Effect of AHL Effect of IPTG Preliminary assays
Effect of AHL
Objective:
By testing how AHL activates lux-lac hybrid promoter, parameters(n2,K2) should be determined.
===Samples: === in DH5 alpha
placIQ(constitutive promoter) + GFP (Pos. con.) :[http://partsregistry.org/Part:BBa_J54201 BBa_J54201]
No promoter + GFP (Neg. con.) :[http://partsregistry.org/Part:BBa_J54102 BBa_J54102]
Lux lac hybrid promoter + GFP :[http://partsregistry.org/Part:BBa_I751900 BBa_I751900]
Procedure:
prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (+ Amp and 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
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
(*The same amount of DMSO, solvent for AHL, was added to each sample.)
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement
Result & Conclusion:
Fluorescence intensity (arbitrary unit, a.u.) as a function of the concentration of AHL was determined.
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.
n2 = 2.08 (-)
K2 = 4.05 (μM)
