Difference between revisions of "Part:BBa K608009"

Latest revision as of 22:49, 21 September 2011

Strong promoter with weak RBS and GFP

Strong promoter from the constitutive promoter family combined with a weak RBS (PR3) and tagged with GFP to quantify the gene expression. The GFP fluorescence intensity 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 concentration. 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 promoter and RBS

Promoter and RBS:
PR1: strong Promoter (J23104) strong RBS (B0034)
PR2: strong Promoter (J23104) medium RBS (B0032)
PR3: strong Promoter (J23104) weak RBS (B0031)
PR4: medium Promoter (J23110) strong RBS (B0034)
PR5: medium Promoter (J23110) medium RBS (B0032)
PR6: medium Promoter (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 PR3 has the lowest expression of GFP. The fluorescence intensity of GFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promoter 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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K608009 short</partinfo>
-Strong promotor from the constitutive promotor family combined with a weak RBS and tagged with GFP to quantify the gene expression.
+Strong promoter from the constitutive promoter family combined with a weak RBS (PR3) and tagged with GFP to quantify the gene expression.
-The GFP fluorescence was measured with a plate reader:<br/>
+The GFP fluorescence intensity 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
+We measured the protein concentration with the bradford-assay. This is a method to determine the total protein concentration. 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.
-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-PR3.jpg|left|thumb|400px|GFP fluorescence intensity dependent on the strenght of promotor and RBS]]
+[[Image:Freiburg2011_GFP-PR3.jpg|left|thumb|400px|GFP fluorescence intensity dependent on the strenght of promoter and RBS]]
-'''Promotor and RBS:'''<br/>
+'''Promoter and RBS:'''<br/>
-PR1:  strong Promotor (J23104) strong RBS (B0034)<br/>
+PR1:  strong Promoter (J23104) strong RBS (B0034)<br/>
-PR2: strong Promotor (J23104) medium RBS (B0032)<br/>
+PR2: strong Promoter (J23104) medium RBS (B0032)<br/>
-'''PR3: strong Promotor (J23104) weak RBS (B0031)'''<br/>
+'''PR3: strong Promoter (J23104) weak RBS (B0031)'''<br/>
-PR4: medium Promotor (J23110) strong RBS (B0034)<br/>
+PR4: medium Promoter (J23110) strong RBS (B0034)<br/>
-PR5: medium Promotor (J23110) medium RBS (B0032)<br/>
+PR5: medium Promoter (J23110) medium RBS (B0032)<br/>
-PR6: medium Promotor (J23110) weak RBS (B0031)<br/>
+PR6: medium Promoter (J23110) weak RBS (B0031)<br/>
 {|cellpadding="10" cellspacing="0" border="1"
@@ Line 49: / Line 48: @@
 <br/>
 <br/>
-The results of this test show that PR3 has the lowest expression of GFP. 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.
+The results of this test show that PR3 has the lowest expression of GFP. The fluorescence intensity of GFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promoter 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.