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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
| | <partinfo>BBa_I751501 SequenceAndFeatures</partinfo> | | <partinfo>BBa_I751501 SequenceAndFeatures</partinfo> |
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| − | ===Usage and Biology===
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| − | The characteristics of this hybrid promoter (Lux-lac hybrid promoter) have been determined though [http://parts.mit.edu/igem07/index.php/Tokyo/Works/Assay0| Assay 0]. The results are shown in Fig.1.
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| − | '''The newly devised hybrid promoter is activated by AHL and repressed by LacI.'''
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| − | <br>In the present project, this hybrid promoter has been tested on the [[Part:BBa_I751100|BBa_I751100. (J54140del Pr)]]
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| − | [[Image : Hybridgraph.JPG|thumb|300px| '''Fig.1: The result of assay0''' <br>Only in the presence of AHL and in the absence of lacI, the lux lac hybrid promoter is activated significantly.|right]]
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_I751501 parameters</partinfo>
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| − | see [http://parts.mit.edu/igem07/index.php/Tokyo/Hill_function_fitting| details]
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| − | == [[Tokyo/AHL assay|1.AHL assay]] ==
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| − | <br>[[Image:hill AHL.JPG|thumb|210px| '''Fig. 2 Effect of AHL on hybrid promoter''' Enhanced fluorescence intensity represents up-regulation of LuxR via AHL. ]]
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| − | '''Purpose'''
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| − | <br>check how AHL activates lux-lac hybrid promoter.
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| − | <br>These parameters(n2,K2) is decided.
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| − | '''Result & Conclusion'''
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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>
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| − | ====⇒see more [http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay| details]====
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| − | == [[Tokyo/IPTG assay|2.IPTG assay]] ==
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| − | [[Image:hill IPTG.JPG|thumb|210px|'''Fig.3 The result of IPTG assay''' Fluorescence intensity as a function of the concentration of IPTG was determined. ]]
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| − | '''Purpose'''
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| − | <br>check how LacI represses this hybrid promoter. In order to adjust LacI repression, IPTG was added.
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| − | <br>These parameters(n3,K3) is decided.
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| − | '''Result & Conclusion'''
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| − | <br>
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| − | The concentration of IPTG dose-denpendently increased GFP fluorescence. It indicates that the hybrid promoter in the cells expressing LacI became increasingly strengthened by decline of repression by LacI. The activation graph in Fig. 3, the characteristics of the hybrid promoter expressed in Hill function is determined.
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| − | <br>
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| − | ====⇒see more [http://parts.mit.edu/igem07/index.php/Tokyo/IPTG_assay| details]====
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| − | <br><br>
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| − | We have determined Hill coefficients, coefficients of repression and activation of AHL and LacI（n2,n3,k2,k3) as follows.
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| − | <br>'''n2 = 2.08 (-)'''
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| − | <br>'''K2 = 4.05 (μM)'''
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| − | <br>'''n3 = 2.47 (-)'''
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| − | <br>'''K3 = 0.295 (μM)'''
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| − | <br>see [http://parts.mit.edu/igem07/index.php/Tokyo/Hill_function_fitting| formulations]
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