Part:BBa_K747096:Experience

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Characterization by UT-Tokyo iGEM 2014

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.

Figure 1 (a) The activity of BBa_K747096GFP was activated by 501nm excitation laser. Measured when OD600 is 0.9~1.1.

Figure 1 (b) The activity of BBa_K747096GFP was activated by 501nm excitation laser. Measured after cultured O/N.

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.

Figure 2 (a) Real-time measurement of BBa_K747096. Activity of GFP (activated by 488nm excitation laser).

Figure 2 (b) Activity of GFP (activated by 488nm excitation laser).

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

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.

Table 1 : Fluorescence / OD

	K747096	J23101
t=4h	92.62	13.14
t=6h	99.37	19.86

From Table1, we can get RPU of BBa_K747096: RPU = 5.8

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

Characterization by BIOSINT_Mexico iGEM 2014

CMV promoter fused with YFP by BIOSINT_Mexico iGEM 2014

As a part of the project from 2014_BIOSINT_Mexico , we fused the promoter PCMV (BBa_K747096) to a YFP reporter (BBa_E0030).

In order to measure its expression, we culture transformed E.coli in agar plates and measured the fluorescense intensity every 45 minutes. After that, we obtained the following data. [Fig.1]

Figure 1 YFP intensity every 45 minutes.

We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:

Figure 2 Media YFP intensity every 45 minutes.

We analysed the data and using the Wolfram Mathematica software, we obtained the following equation that fits the data. If we plot this equation, we obtain:

Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.

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