Difference between revisions of "Part:BBa I751501"
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== [[Tokyo/AHL assay|1.AHL assay]] ==
 
== [[Tokyo/AHL assay|1.AHL assay]] ==
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<br>[[Image:hill AHL.JPG|thumb|210px| '''Fig. 3 Effect of AHL on hybrid promoter''' Enhanced fluorescence intensity represents up-regulation of LuxR via AHL. ]]
+
<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. ]]
 
'''Purpose'''
 
'''Purpose'''
 
<br>check how AHL activates lux-lac hybrid promoter.
 
<br>check how AHL activates lux-lac hybrid promoter.

Revision as of 23:11, 26 October 2007

plux-cI hybrid promoter

sense TWO INPUTS, activation by AHL and repression by Lambda cI.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

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. The newly devised hybrid promoter is activated by AHL and repressed by LacI.
In the present project, this hybrid promoter has been tested on the BBa_I751100. (J54140del Pr)


Fig.1: The result of assay0
Only in the presence of AHL and in the absence of lacI, the lux lac hybrid promoter is activated significantly.













Functional Parameters

negative_regulators1
positive_regulators1

see [http://parts.mit.edu/igem07/index.php/Tokyo/Hill_function_fitting| details]

1.AHL assay


Fig. 2 Effect of AHL on hybrid promoter Enhanced fluorescence intensity represents up-regulation of LuxR via AHL.

Purpose
check how AHL activates lux-lac hybrid promoter.
These parameters(n2,K2) is decided.

Result & Conclusion
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.


⇒see more [http://parts.mit.edu/igem07/index.php/Tokyo/AHL_assay| details]

2.IPTG assay

Fig.3 The result of IPTG assay Fluorescence intensity as a function of the concentration of IPTG was determined.

Purpose
check how LacI represses this hybrid promoter. In order to adjust LacI repression, IPTG was added.
These parameters(n3,K3) is decided.

Result & Conclusion
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.

⇒see more [http://parts.mit.edu/igem07/index.php/Tokyo/IPTG_assay| details]



We have determined Hill coefficients, coefficients of repression and activation of AHL and LacI（n2,n3,k2,k3) as follows.
n2 = 2.08 (-)
K2 = 4.05 (μM)
n3 = 2.47 (-)
K3 = 0.295 (μM)
see [http://parts.mit.edu/igem07/index.php/Tokyo/Hill_function_fitting| formulations]