Difference between revisions of "Part:BBa K1119008"
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<partinfo>BBa_K1119008 short</partinfo>
 
<partinfo>BBa_K1119008 short</partinfo>
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Composite part for characterization of CMV promoter. 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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This is a mammalian GFP generator used for characterization of our CMV promoter ([[Part:BBa_K1119006|BBa_K1119006]]). This GFP generator contains the CMV promoter sequence with GFP reporter in RFC25 format([[Part:BBa_K648013|BBa_K648013]]) and hGH polyA terminator ([[Part:BBa_K404108|BBa_K404108]]).
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This GFP generator was then transfected into HEK293FT cells and <i>in vivo</i> green fluorescence signal was observed under confocal microscope.
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pEGFP-N1 (Clontech) served as the positive control. It contains the CMV promoter and EGFP reporter. A GFP generator that does not contain the CMV promoter was used as the negative control.  
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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 gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals.]]
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===Improved Part===
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iGEM Technion 2017 improved this part to <partinfo>BBa_K2520006</partinfo>. This improved device contains a TRE promoter instead of the original CMV, to allow for inducible expression of GFP within mammalian cells.
  
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CMV promoter characterization construct composes of CMV promoter, Green Fluorescence Protein (GFP) and hGH polyadenylation sequence (hGH pA) in pSB1C3 backbone.(BBa_K1119006 – BBa_K648013 – BBa_K404108 – pSB1C3)
 
  
  

Latest revision as of 17:07, 30 October 2017

CMV promoter - GFP - hGH polyA tail

This is a mammalian GFP generator used for characterization of our CMV promoter (BBa_K1119006). This GFP generator contains the CMV promoter sequence with GFP reporter in RFC25 format(BBa_K648013) and hGH polyA terminator (BBa_K404108).

This GFP generator was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.

pEGFP-N1 (Clontech) served as the positive control. It contains the CMV promoter and EGFP reporter. A GFP generator that does not contain the CMV promoter was used as the negative control.

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 gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals.

Improved Part

iGEM Technion 2017 improved this part to BBa_K2520006. This improved device contains a TRE promoter instead of the original CMV, to allow for inducible expression of GFP within mammalian cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 628
    Illegal AgeI site found at 1369
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1772
    Illegal BsaI.rc site found at 1274