Team:UT-Tokyo/CTCD/Content/Modeling

From 2014.igem.org

<img src ="SubCTC_BBaK747096.png" class = "contTitle" />

Characterization of BBa_K747096

During DNA assembly, we found that this promoter was activated in Escherichia coli. Then we decided to characterize this promoter activity by comparing with constitutive promoter (BBa_J23101) using GFP (BBa_E0040) (Figure 1).
As seen in Figure 1, this promoter shows weaker activity than BBa_J23101.

<img src = "Graph%28assay5%29_01wiki.png" class = "figure" style="width:400px; float:left;" /> <img src = "Graph%28assay5%29_maxwiki.png" class = "figure" style="width:400px; float:left;padding-left:0px;" /> <legend style="clear:both">Fig. 1The activity of BBa_K747096GFP was activated by 501nm excitation laser.</legend> <legend style="width:300px;float:left;margin-left:120px;margin-right:100px;">a)Measured when OD600 is 0.9~1.1.</legend> <legend style="width:300px;float:left;margin-left:0px;margin-right:0px;">b)Measured after cultured O/N.</legend>

In order to calculate RPU (relative promoter unit [1]), we performed real-time measurement of GFP fluorescence (Figure 2). We measured activity of GFP and OD600, which are necessary for calculation of RPU.

<img src = "Assay5_GFP_wiki.png" class = "figure" style="width:400px; float:left;" /> <img src = "Assay5_OD600_wiki.png" class = "figure" style="width:400px; float:left;padding-left:0px;" /> <legend style="clear:both;margin-left:250px;">Fig. 2Real-time measurement of BBa_K747096</legend> <legend style="width:400px;float:left;margin-left:60px;margin-right:150px;">a)Activity of GFP (activated by 488nm excitation laser).</legend> <legend style="width:200px;float:left;margin-left:0px;margin-right:0px;">b)OD600.</legend>

We calculated RPU(Relative Promoter Unit) of BBa_K747096 from the Fig. 2 using the equation (1).

<img src="Cocoa_equation_1.png" class="math" height="60px" style="clear:both;"/>

This equation can be used only when Fluorescence per cell is in steady state. In this experiment, this condition was fulfilled as shown in Table1.

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Table 1 : Fluorescence / OD
K747096J23101
t=4h92.6213.14
t=6h99.3719.86

From Table1, we can get RPU of BBa_K747096:

RPU = 5.8

[1]:https://2010.igem.org/Team:Kyoto/LearnMore#Relative_Promoter_Unit_.28RPU.29

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