# Part:BBa_K747096:Experience

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### Applications of BBa_K747096

## 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.

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.

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.

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]

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

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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