Tokyo/Works/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]]
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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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<br><br>
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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 LacI-IPTG]]　　[[Tokyo/Preliminary assays |Preliminary assays]]
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=1.Important assay =
 
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==[[Tokyo/Hill function fitting |Hill function fitting ]] ==
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==[[Tokyo/Hill function fitting |Objective of this assay (Hill function fitting) ]] ==
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新しく作ったハイブリッドプロモーターに関してはデータがないので，実験によりパラメータを求める．
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<!--新しく作ったハイブリッドプロモーターに関してはデータがないので，実験によりパラメータを求める．
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ヒル関数をfittingさせることにより，n2,n3,K2,K3が求まる.
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ヒル関数をfittingさせることにより，n2,n3,K2,K3が求まる.-->
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We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined n2, n3, K2, and K3.  
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We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined '''n2, n3, K2, and K3'''.  
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<br>[[Image:expression6-1.JPG|300px|]]
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<br>[[Image:siki1.JPG|300px|none|]]
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<br>[[Image:expression6-2.JPG|340px|]]
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<br>[[Image:expression6-10-2.JPG|320px|left|]][[Image:siki2.JPG|400px|none|]]
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<br>
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<br>
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==== ==> see more [[Tokyo/Hill function fitting|details]]====
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導出に関してはfor more detail.
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<!--== Assays for hybrid promoter ==
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== Assays for hybrid promoter ==
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These assays were done  
These assays were done  
<br>-to determine the characteristics of our hybrid promoter.
<br>-to determine the characteristics of our hybrid promoter.
<br>The data from here were used for computational analyses and simulations.
<br>The data from here were used for computational analyses and simulations.
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-->
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== [[Tokyo/Repression and activation check with LacI and AHL|Repression and activation check with LacI and AHL]] ==
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<br>
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====Purpose====
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== [[Tokyo/AHL assay|Effect of AHL]] ==
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<br>check if AHL activates lux-lac hybrid promoter  
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<br>[[Image:hill AHL.JPG|thumb|230px|'''Fig. 1 Effect of AHL on hybrid promoter''' Enhanced fluorescence intensity represents up-regulation of LuxR via AHL.|left]]
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<br>check if lacI represses lux-lac hybrid promoter
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'''Objective'''
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By testing how AHL activates lux-lac hybrid promoter,  parameters(n2,K2) should be determined.
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====Result & Conclusion====
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<br><br>
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<br>AND gate by AHL & IPTG
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'''Result & Conclusion'''
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<br>Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG.
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<br>GFP fluorescence increased in AHL-dose dependent manner, indicating that the hybrid promoter is enhanced by LuxR via AHL. Taken together with the graph shown in Fig.1 and formulation of Hill function fitting described above , the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), are determined.  
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== [[Tokyo/IPTG assay|IPTG assay]] ==
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====Purpose====
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<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.
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<br> The order of the concentration is used for more detailed assay with narrower range of the IPTG concentration.
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====Result & Conclusion====
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== [[Tokyo/Activation check by cell-produced AHL |Activation check by cell-produced AHL ]] ==
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==== Purpose ====
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To check if worker E. coli (Sender) can produce enough AHL for our model to work by using different copy numbers of plasmids.
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<br>
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==== ==> see more [[Tokyo/AHL assay|details]]====
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<br>
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<br>
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<br>
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====Result & Conclusion====
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== [[Tokyo/IPTG assay|Effect of LacI: IPTG assay]] ==
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[[Image:AHL_assay_endogenous_Results.jpg]]
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[[Image:hill IPTG.JPG|thumb|215px|'''Fig. 2 Effect of LacI on hybrid promoter''' Enhanced fluorescence intensity represents down-regulation of LacI repression via IPTG.|left]]
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'''Objective'''
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By testing how LacI represses the lux-lac hybrid promoter, parameters(n3,K3) should be decided. In order to adjust effective concentration of LacI, IPTG was added.
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<br><br>
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'''Result & Conclusion'''
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GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in Fig. 2 and the formulation of Hill function fitting described above, the characteristics of the hybrid promoter expressed in Hill function was determined.
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==== ==> see more [[Tokyo/IPTG assay|details]]====
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<br>
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<br>
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<br>
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<br>Not only high copy number plasmid pSB1, also low copy number plasmid pSB4 and pBR produced enough AHL to activate the LacI hybrid promoter in other cells. Especially, pBR remarkably produced AHL in the present experiment.
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==Appendix: [[Tokyo/Preliminary assays |Preliminary assays ]]==
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We conducted preliminary assays as practice experiments and check of materials and methods.
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<br>
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==== ==> See more [[Tokyo/Preliminary assays |details]]====
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=2.Preliminary assays=
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== ==
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'''We conducted preliminary assays to practice experiments and check if the materials and methods worked properly.'''
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[[Tokyo/Works/Formulation |Before(Formulation)]]　<<　[[Tokyo/Works/Assay |Assay1]]　>>　[[Tokyo/Hill function fitting|Assay1 contents]]　>>>>　[[Tokyo/Works/Simulation |Simulation]]　　
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<br>[[Tokyo/Preliminary assays |See more details]]
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Latest revision as of 04:53, 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 LacI-IPTG　　Preliminary assays


Objective of this assay (Hill function fitting)

We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined n2, n3, K2, and K3.


Siki1.JPG

Expression6-10-2.JPG
Siki2.JPG



==> see more details


Effect of AHL


Fig. 1 Effect of AHL on hybrid promoter Enhanced fluorescence intensity represents up-regulation of LuxR via AHL.

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

Result & Conclusion
GFP fluorescence increased in AHL-dose dependent manner, indicating that the hybrid promoter is enhanced by LuxR via AHL. Taken together with the graph shown in Fig.1 and formulation of Hill function fitting described above , the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), are determined.


==> see more details




Effect of LacI: IPTG assay

Fig. 2 Effect of LacI on hybrid promoter Enhanced fluorescence intensity represents down-regulation of LacI repression via IPTG.

Objective By testing how LacI represses the lux-lac hybrid promoter, parameters(n3,K3) should be decided. In order to adjust effective concentration of LacI, IPTG was added.

Result & Conclusion GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in Fig. 2 and the formulation of Hill function fitting described above, the characteristics of the hybrid promoter expressed in Hill function was determined.

==> see more details






Appendix: Preliminary assays

We conducted preliminary assays as practice experiments and check of materials and methods.

==> See more details

Before(Formulation)　<<　Assay1　>>　Assay1 contents　>>>>　Simulation　　

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