Difference between revisions of "Part:BBa K1119006"
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<partinfo>BBa_K1119006 short</partinfo>
 
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CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter that we used to express glyoxylate genes in mammalian cell. Our objective in characterizing this promoter is to test if the CMV promoter drives the expression of green fluorescence protein (GFP) in mammalian HEK293FT cell line.  
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CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.  
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The partsregistry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. In addition, there are five twins of this CMV promoter, including BBa_I712004 was submitted by Ljubljana 2007 team and characterized by Heidelberg 2009 team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. Although there were many uses of this promoter, our team decided to build a reliable CMV promoter for our constitutive glyoxylate shunt construct. 
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===Characterization===
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In our characterization, the coding sequence of CMV promoter was assembled with GFP reporter ([[Part:BBa_K648013|BBa_K648013]]) and hGH polyA terminator ([[Part:BBa_K404108|BBa_K404108]]) using Freiburg’s RFC25 format.
  
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The partsregistry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. In addition, the twin of this CMV promoter BBa_I712004 was submitted by Ljubljana 2007 team and characterized by Heidelberg 2009 team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. Although there were many uses of this promoter, our team decided to build a reliable CMV promoter for our constitutive glyoxylate shunt construct.  
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The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.
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The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator that does not contain the CMV promoter.  
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The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.
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[[File:Final CMV annotated no ABC.jpg|600px|thumb|center|'''Figure 1. CMV promoter drives expression of GFP.''' HEK cells transfected with pCMV-GFP expressed GFP. Signal. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the negative control GFP without any promoter was not expressed.   ]]
  
 
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Revision as of 10:32, 26 September 2013

CMV promoter

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

The partsregistry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. In addition, there are five twins of this CMV promoter, including BBa_I712004 was submitted by Ljubljana 2007 team and characterized by Heidelberg 2009 team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. Although there were many uses of this promoter, our team decided to build a reliable CMV promoter for our constitutive glyoxylate shunt construct.

Characterization

In our characterization, the coding sequence of CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108) using Freiburg’s RFC25 format.

The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.

The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator that does not contain the CMV promoter.

The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1. CMV promoter drives expression of GFP. HEK cells transfected with pCMV-GFP expressed GFP. Signal. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the negative control GFP without any promoter was not expressed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]