Difference between revisions of "Part:BBa K1119006"

(→‎Issues with Previously Submitted CMV Promoters)
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===Issues with Previously Submitted CMV Promoters===
 
===Issues with Previously Submitted CMV Promoters===
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The Part Registry contains CMV promoter ([[Part:BBa_J52034|BBa_J52034]]) submitted by Slovenia 2006 team and [[Part:BBa_J52034:Experience#Characterisation|characterized by DTU-Denamrk 2011 team]]. In addition, there are five twins of this CMV promoter, including [[Part:BBa_I712004|BBa_I712004]], which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength|characterized by Heidelberg 2009] team using [[Part:BBa_K203100|BBa_K203100]] pSMB_MEASURE (Promoter measurement plasmid). However, according to the user reviews, team LMU-Munich 2010 stated that [[https://parts.igem.org/Part:BBa_J52034:Experience#User%20Reviews|this part is not a CMV promoter but rather a long version of ''lacI'' gene for prokaryotes]]. Since we could identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.   
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The Part Registry contains CMV promoter ([[Part:BBa_J52034|BBa_J52034]]) submitted by Slovenia 2006 team and [[Part:BBa_J52034:Experience#Characterisation|characterized by DTU-Denamrk 2011 team]]. However, according to the user reviews, team LMU-Munich 2010 stated that [[Part:BBa_J52034:Experience#User%20Reviews|this part is not a CMV promoter but rather a long version of ''lacI'' gene for prokaryotes]]. In addition, there are five twins of this CMV promoter, including [[Part:BBa_I712004|BBa_I712004]], which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength|characterized by Heidelberg 2009] team using [[Part:BBa_K203100|BBa_K203100]] pSMB_MEASURE (Promoter measurement plasmid). Since we could identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.   
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<span style="color:red">Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.</span>
 
<span style="color:red">Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.</span>
  

Revision as of 15:22, 26 September 2013

CMV promoter

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

Issues with Previously Submitted CMV Promoters

The Part Registry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. 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. In addition, there are five twins of this CMV promoter, including BBa_I712004, which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength|characterized by Heidelberg 2009] team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). Since we could identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.

Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.

Characterization

In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108).

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 (BBa_K648013) 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]