Tokyo/AHL assay

From 2007.igem.org

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__NOTOC__
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<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]]
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<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]]
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[[Tokyo/Hill function fitting |Purpose of this assay]]　　[[Tokyo/AHL assay|1.AHL assay]]　　[[Tokyo/IPTG assay|2.IPTG assay]]　　[[Tokyo/Preliminary assays |Preliminary assays]]
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[[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]]
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== AHL assay ==
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== Effect of AHL ==
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===Objective: ===
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<br>By testing how AHL activates lux-lac hybrid promoter, parameters(n2,K2) should be determined.
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===Purpose: ===
 
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<br>To check how AHL activates the newly devised lux-lac hybrid promoter
 
<!--<br>check how lacI represses lux-lac hybrid promoter-->
<!--<br>check how lacI represses lux-lac hybrid promoter-->
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===Samples: ===
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===Samples: === in DH5 alpha
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<br>placQI + GFP (constitutive promoter) (Pos. con.)
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<br>placIQ(constitutive promoter) + GFP (Pos. con.) :[http://partsregistry.org/Part:BBa_J54201 BBa_J54201]
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<br>No promoter + GFP (Neg. con.)
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<br>No promoter + GFP (Neg. con.) :[http://partsregistry.org/Part:BBa_J54102 BBa_J54102]
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<br>Lux lac hybrid promoter + GFP
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<br>Lux lac hybrid promoter + GFP :[http://partsregistry.org/Part:BBa_I751900 BBa_I751900]
<!--<br>hybrid promoter plasmid with pTrc99A  
<!--<br>hybrid promoter plasmid with pTrc99A  
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===Result & Conclusion: ===
===Result & Conclusion: ===
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[[Image:hill AHL.JPG|thumb|300px| '''Fig.2: '''Result of AHL assay]]
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[[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. ]]
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<br>As the concentration of AHL increases, GFP fluorescence increased, indicating 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.
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<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.
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<br>'''n2 = 2.08 (-)'''
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<br>'''K2 = 4.05 (μM)'''
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<!--<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 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:

Fig.1: Activated by externally added AHL, cells had accumulated GFP, increasing fluorescence intensity.


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:

Fig.2: Result of AHL assay
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)


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