Difference between revisions of "Part:BBa K747096:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K747096===
 
===Applications of BBa_K747096===
  
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As part of the iGEM team BIOSINT_Mexico for 2014, e fused the promoter PCMV (BBa_K747096) to a YFP reporter (BBa_E0030).  
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==Characterization by UT-Tokyo iGEM 2014==
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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.
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[[File:Graph(assay5)_01wiki.png|400px|thumb|left|center|'''Figure 1 (a)''' The activity of BBa_K747096GFP was activated by 501nm excitation laser. Measured when OD600 is 0.9~1.1.]]
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[[File:Graph(assay5)_maxwiki.png|400px|thumb|left|center|'''Figure 1 (b)''' The activity of BBa_K747096GFP was activated by 501nm excitation laser. Measured after cultured O/N.]]
  
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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]
 
  
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:::[[File:1res.png|150px|thumb|left|'''Figure 1''' YFP intensity every 45 minutes.]]
 
  
  
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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.
  
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[[File:assay5_GFP_wiki.png|400px|thumb|left|center|'''Figure 2 (a)''' Real-time measurement of BBa_K747096. Activity of GFP (activated by 488nm excitation laser).]]
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[[File:assay5_OD600_wiki.png|400px|thumb|left|center|'''Figure 2 (b)''' Activity of GFP (activated by 488nm excitation laser).]]
  
  
  
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We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:
 
  
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::::::[[File:2res.png|300px|thumb|left|'''Figure 2''' Media YFP intensity every 45 minutes.]]
 
  
  
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We calculated RPU(Relative Promoter Unit) of BBa_K747096 from the Fig. 2 using the equation (1).
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[[File:cocoa_equation_1.png|600px]]
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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 border="1" width="850" align="center" style="background-color:#FFF;"> <caption>Table 1 : Fluorescence / OD</caption>
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<tr bgcolor="#cccccc">
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<th></th><th>K747096</th><th>J23101</th></tr>
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<tr align="center"><td>t=4h</td><td>92.62</td><td>13.14</td></tr>
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<tr align="center"><td>t=6h</td><td>99.37</td><td>19.86</td></tr>
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</table>
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From Table1, we can get RPU of BBa_K747096:
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RPU = 5.8
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[1]:http://2010.igem.org/Team:Kyoto/LearnMore#Relative_Promoter_Unit_.28RPU.29
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==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]
 +
 
 +
[[File:6res.png|150px|thumb|left|'''Figure 1''' YFP intensity every 45 minutes.]]
 +
 
 +
We made three repetitions, so in order to model the equation, we obtained the mean measure, therefore:
 +
 
 +
::::::[[File:2res.png|300px|thumb|left|'''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.
 
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:
 
If we plot this equation, we obtain:
 +
  
 
[[File:Model1res.png|400px|center|]]
 
[[File:Model1res.png|400px|center|]]
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Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.  
 
Where we can see that the intensity of YFP grows directly proportional to the time and concentration of the molecule.  
  
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::::::[[File:4res.png|600px|left|center|'''Figure 3''' Intensity YFP vs time.]]
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::::::[[File:1res.png|600px|left|center|'''Figure 3''' Intensity YFP vs time.]]
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<br style="clear:both;"/>
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 02:00, 2 November 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

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).

Cocoa equation 1.png

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
K747096J23101
t=4h92.6213.14
t=6h99.3719.86

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:


Model1res.png

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


Figure 3 Intensity YFP vs time.


User Reviews

UNIQbea2709761150b57-partinfo-00000000-QINU UNIQbea2709761150b57-partinfo-00000001-QINU