Tokyo/AHL assay
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__NOTOC__ | __NOTOC__ | ||
| - | + | <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>[[Tokyo/Works|Works top]] | + | <br><br> |
| + | [[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]] | ||
| - | == AHL | + | == Effect of AHL == |
| - | === | + | ===Objective: === |
| - | <br> | + | <br>By testing how AHL activates lux-lac hybrid promoter, parameters(n2,K2) should be determined. |
| - | + | ||
| - | ===Samples: === | + | <!--<br>check how lacI represses lux-lac hybrid promoter--> |
| - | <br>hybrid promoter plasmid with pTrc99A | + | |
| + | ===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>hybrod promoter plasmid with pBR322 | ||
<br>luxR plasmid with pTrc99A | <br>luxR plasmid with pTrc99A | ||
<br>luxR --- AHL-dependent activation confirmed | <br>luxR --- AHL-dependent activation confirmed | ||
<br>placIq on promter-less GFP in DH5a (for pos. con.) | <br>placIq on promter-less GFP in DH5a (for pos. con.) | ||
| - | <br>promoter-less GFP in DH5a (for neg. con.) | + | <br>promoter-less GFP in DH5a (for neg. con.)--> |
===Procedure: === | ===Procedure: === | ||
| - | [[Image:AHL Assay2.JPG|thumb|200px| '''Fig.1: ''' | + | [[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>prepare overnight culture for each sample | ||
<br>make fresh culture | <br>make fresh culture | ||
| - | <br>take 3 ul of the overinight culture into 3 ml of LB (Amp and | + | <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>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>add AHL & IPTG solution | ||
| - | <br> | + | <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> | + | <br>(*The same amount of DMSO, solvent for AHL, was added to each sample.) |
<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: === | ||
| - | + | [[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>AND gate by AHL & IPTG | + | <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>Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG. | + | |
| + | <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.--> | ||
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)
