Difference between revisions of "Part:BBa K238013:Experience"

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===User Reviews===
 
===User Reviews===
 
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<partinfo>BBa_K238013</partinfo>
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<I>ETH Zürich iGEM 2012</I>
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'''ETH Zürich iGEM 2012'''
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We used this part as a blue light inducible repression system. Fortunately the research team of Natalie Tschowri at FU Berlin [http://www.ncbi.nlm.nih.gov/pubmed/22783906 recently published] new results including a DNA footprint for the inverted repeats in the YcgZ promoter. We compared the inverted repeats in their research to the ones indicated on <partinfo>BBa_K238013</partinfo> and we found them to be different.
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[[Image:Tschowri2012_DNAFootprint.png|620px|middle|thumb|A) The DNA footprint done in the research of [http://www.ncbi.nlm.nih.gov/pubmed/22783906 Natalia Tschowri 2012] that shows the binding regions / inverted repeats for BluR (YcgE). B) Shift Assay: Point mutations in the determined binding regions drastically decrease DNA binding of BluR (YcgE).]]
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The clear indication of the inverted repeats allowed us to improve this part by adding additional operator regions. The '''improved part can be found here''': <partinfo>BBa_K909010</partinfo>
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<I>Glasgow iGEM 2011</I>
 
<I>Glasgow iGEM 2011</I>
 
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'''Glasgow iGEM 2011'''
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We coupled the bluf promoter to YFP and measured intensity after induction with white light. We found that expression showed a 2.4* increase in fold induction when compared with darkness as a control.
 
We coupled the bluf promoter to YFP and measured intensity after induction with white light. We found that expression showed a 2.4* increase in fold induction when compared with darkness as a control.
  
 
The promoter demonstrated leaky expression with no light induction.
 
The promoter demonstrated leaky expression with no light induction.
  
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[[Image:Gla_Bluf plus YFP.jpg]]
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[[Image:Gla_YFP_Results.jpg]]
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The image above demonstrates expression of YFP ligated to an RBS and the bluf promoter. The images are taken from two samples, one which was grown in darkness and the other grown with light present. The samples were shaken overnight at room temperature and the intensity of expression was measured. (See graph below)
 
The image above demonstrates expression of YFP ligated to an RBS and the bluf promoter. The images are taken from two samples, one which was grown in darkness and the other grown with light present. The samples were shaken overnight at room temperature and the intensity of expression was measured. (See graph below)
  
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Scale = 15 microns.  
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Scale = 10 microns.  
  
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Fluorescence filter : 515-565nm  
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1st Fluorescence filter : 515-565nm (left column)
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2nd Fluorescence filter : 470/20 nm (right column)
  
 
[[Image:Bluf graph.jpg]]
 
[[Image:Bluf graph.jpg]]
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The graph clearly shows that expression has been significantly induced by white light for expression with the bluf domain. When averaged out, the total induction shows an increase in YFP fluorescence by a 2.4 fold level, however, the promoter does exhibit some leaky expression.
 
The graph clearly shows that expression has been significantly induced by white light for expression with the bluf domain. When averaged out, the total induction shows an increase in YFP fluorescence by a 2.4 fold level, however, the promoter does exhibit some leaky expression.
  
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This was the only light-induced promoter, out of the 3 that the Glasgow iGEM 2011 team trialled, which demonstrated any measurable increase in expression.
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The construct used to test fluorescence is available here (https://parts.igem.org/wiki/index.php?title=Part:BBa_K660601)
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Latest revision as of 15:39, 26 September 2012

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_K238013

User Reviews

UNIQa6bc807729814911-partinfo-00000000-QINU

BBa_K238013

ETH Zürich iGEM 2012

ETH Zürich iGEM 2012

We used this part as a blue light inducible repression system. Fortunately the research team of Natalie Tschowri at FU Berlin [http://www.ncbi.nlm.nih.gov/pubmed/22783906 recently published] new results including a DNA footprint for the inverted repeats in the YcgZ promoter. We compared the inverted repeats in their research to the ones indicated on BBa_K238013 and we found them to be different.

A) The DNA footprint done in the research of [http://www.ncbi.nlm.nih.gov/pubmed/22783906 Natalia Tschowri 2012] that shows the binding regions / inverted repeats for BluR (YcgE). B) Shift Assay: Point mutations in the determined binding regions drastically decrease DNA binding of BluR (YcgE).

The clear indication of the inverted repeats allowed us to improve this part by adding additional operator regions. The improved part can be found here: BBa_K909010


;

BBa_K238013 Glasgow iGEM 2011

Glasgow iGEM 2011

We coupled the bluf promoter to YFP and measured intensity after induction with white light. We found that expression showed a 2.4* increase in fold induction when compared with darkness as a control.

The promoter demonstrated leaky expression with no light induction.

Gla YFP Results.jpg

The image above demonstrates expression of YFP ligated to an RBS and the bluf promoter. The images are taken from two samples, one which was grown in darkness and the other grown with light present. The samples were shaken overnight at room temperature and the intensity of expression was measured. (See graph below)

Scale = 10 microns.

1st Fluorescence filter : 515-565nm (left column)

2nd Fluorescence filter : 470/20 nm (right column)

Bluf graph.jpg

The graph clearly shows that expression has been significantly induced by white light for expression with the bluf domain. When averaged out, the total induction shows an increase in YFP fluorescence by a 2.4 fold level, however, the promoter does exhibit some leaky expression.

The construct used to test fluorescence is available here (https://parts.igem.org/wiki/index.php?title=Part:BBa_K660601)

;

UNIQa6bc807729814911-partinfo-00000005-QINU