Difference between revisions of "Part:BBa K608012"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K608012 short</partinfo>
 
<partinfo>BBa_K608012 short</partinfo>
  
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Medium promotor from the constitutive promotor family combined with a weak RBS and tagged with GFP to quantify the gene expression
+
Medium promotor from the constitutive promotor family combined with a weak RBS (PR6) and tagged with GFP to quantify the gene expression.
 +
 
 +
The GFP fluorescence was measured with a plate reader:<br/>
 +
[[Image:Freiburg2011_BSA.jpg|right|350px|thumb|BSA calibration line]]
 +
The fluorescence intensity and protein concentration were measured with the FLUOstar Omega, <br/>
 +
which is a multi-mode microplate reader.
 +
Samples were pipetted into the microplate and analyzed via the plate reader. In this experiment we focused on the protein concentration and the fluorescence intensity of RFP.
 +
We measured the protein concentration with the bradford-assay. This is a method to determine the total protein
 +
To analyze the protein concentration of the samples, Coomassie Brillant Blue was pippeted to each sample. With the binding of the dye to the proteins the color changes from dark red to blue. The more protein in the solution the more Coomassie dye can bind to proteins and the more the color changes into blue. The absorption of bound Coomassie dye is 595nm. The absorbance is proportional with the amount of bound dye. With a series of Bovine Serum Albumin (BSA) measurements the exact protein concentration of the samples can be determined. BSA acts like a “marker” because the concentration of BSA is known and with a linear calibration line the exact protein concentration can be detected.
 +
 
 +
 
 +
GFP served as a reporter of expression. We wanted to know how strong the promoter and RBS activity is. With this reporter gene it was possible to analyze the expression via plate reader. GFP is excited at a wavelength of 509nm and has an emission of 520nm. The plate reader illuminates the samples with a high energy xenon flash lamp. Optical filters or monochromator create the exact wavelength. The more GFP in the sample the higher is the GFP fluorescence intensity. The intensity is collected with the second optical system and is detected with a side window photomultiplier tube.
 +
 
 +
[[Image:Freiburg2011_GFP-PR6.jpg|left|thumb|400px|GFP fluorescence intensity dependent on the strenght of promotor and RBS]]
 +
 
 +
'''Promotor and RBS:'''<br/>
 +
PR1:  strong Promotor (J23104) strong RBS (B0034)<br/>
 +
PR2: strong Promotor (J23104) medium RBS (B0032)<br/>
 +
PR3: strong Promotor (J23104) weak RBS (B0031)<br/>
 +
PR4: medium Promotor (J23110) strong RBS (B0034)<br/>
 +
PR5: medium Promotor (J23110) medium RBS (B0032)<br/>
 +
'''PR6: medium Promotor (J23110) weak RBS (B0031)'''<br/>
 +
 
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|sample
 +
|PR2
 +
|PR3
 +
|PR4
 +
|PR5
 +
|'''PR6'''
 +
|-
 +
|<span style="color:green;">GFP fluorescence intensity</span>
 +
|11378.5
 +
|1445.0
 +
|4596.2
 +
|41221.1
 +
|'''26922.7'''
 +
|-
 +
|factor
 +
|7.9
 +
|1.0
 +
|3.2
 +
|28.5
 +
|'''18.6'''
 +
|}
 +
<br/>
 +
<br/>
 +
<br/>
 +
The results of this test show that PR6 has 18.6 times higher expression of GFP in comparison with with PR3 which has the lowest expression. The fluorescence intensity of GFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promotor and RBS activity with RFP as a reporter and the results deviate from this experiment. So we are looking forward to test this system another time.
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:36, 21 September 2011

Medium promoter with weak RBS and GFP

Medium promotor from the constitutive promotor family combined with a weak RBS (PR6) and tagged with GFP to quantify the gene expression.

The GFP fluorescence was measured with a plate reader:

BSA calibration line

The fluorescence intensity and protein concentration were measured with the FLUOstar Omega,
which is a multi-mode microplate reader. Samples were pipetted into the microplate and analyzed via the plate reader. In this experiment we focused on the protein concentration and the fluorescence intensity of RFP. We measured the protein concentration with the bradford-assay. This is a method to determine the total protein To analyze the protein concentration of the samples, Coomassie Brillant Blue was pippeted to each sample. With the binding of the dye to the proteins the color changes from dark red to blue. The more protein in the solution the more Coomassie dye can bind to proteins and the more the color changes into blue. The absorption of bound Coomassie dye is 595nm. The absorbance is proportional with the amount of bound dye. With a series of Bovine Serum Albumin (BSA) measurements the exact protein concentration of the samples can be determined. BSA acts like a “marker” because the concentration of BSA is known and with a linear calibration line the exact protein concentration can be detected.


GFP served as a reporter of expression. We wanted to know how strong the promoter and RBS activity is. With this reporter gene it was possible to analyze the expression via plate reader. GFP is excited at a wavelength of 509nm and has an emission of 520nm. The plate reader illuminates the samples with a high energy xenon flash lamp. Optical filters or monochromator create the exact wavelength. The more GFP in the sample the higher is the GFP fluorescence intensity. The intensity is collected with the second optical system and is detected with a side window photomultiplier tube.

GFP fluorescence intensity dependent on the strenght of promotor and RBS

Promotor and RBS:
PR1: strong Promotor (J23104) strong RBS (B0034)
PR2: strong Promotor (J23104) medium RBS (B0032)
PR3: strong Promotor (J23104) weak RBS (B0031)
PR4: medium Promotor (J23110) strong RBS (B0034)
PR5: medium Promotor (J23110) medium RBS (B0032)
PR6: medium Promotor (J23110) weak RBS (B0031)

sample PR2 PR3 PR4 PR5 PR6
GFP fluorescence intensity 11378.5 1445.0 4596.2 41221.1 26922.7
factor 7.9 1.0 3.2 28.5 18.6




The results of this test show that PR6 has 18.6 times higher expression of GFP in comparison with with PR3 which has the lowest expression. The fluorescence intensity of GFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promotor and RBS activity with RFP as a reporter and the results deviate from this experiment. So we are looking forward to test this system another time.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 707