Tokyo/IPTG assay

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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]]
<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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[[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]]
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[[Tokyo/Hill function fitting |Objective of this assay]]  [[Tokyo/AHL assay|Effect of AHL]]  [[Tokyo/IPTG assay|Effect of LacI-IPTG]]  [[Tokyo/Preliminary assays |Preliminary assays]]
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== IPTG assay ==
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== LacI-IPTG assay ==
===Objective: ===
===Objective: ===
<br>To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells.
<br>To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells.

Revision as of 20:56, 26 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 LacI-IPTG  Preliminary assays


LacI-IPTG assay

Objective:


To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells.
The order of the concentration is used for more detailed assay with narrower range of the IPTG concentration.

Samples:


A4 pcI([http://partsregistry.org/Part:BBa_I751103 BBa_I751103]) into pTrc99A cell
A4 ΔP([http://partsregistry.org/Part:BBa_I751100 BBa_I751100]) into pTrc99A cell
A4 ΔP + Lux-lac hybrid promter ([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) into pBR322 cell
A4 ΔP + Lux-lac hybrid promter ([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) into pTrc99A cell

Procedure:

Fig.1: As more IPTG is exogenously (externally) added, the fluorescence becomes more intense in the presence of AHL.


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 0.5
add AHL & IPTG solution
[AHL]final (in 3 ml LB culture) = 10 nM
[IPTG]final (in 3 ml LB culture) = 1000, 100, 10, 1, 0.1, 0.01, and 0 mM
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement

Result & Conclusion:

Fig.2: Fluorescence intensity as a function of the concentration of IPTG was determined.

As the concentration of IPTG increases, GFP fluorescence increased, indicating that the hybrid promoter in the LacI expressing cell became increasingly strengthened by decreasing repression by LacI. The activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function is determined.


n3 = 2.47 (-)
K3 = 0.295 (μM)