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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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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The CDS of MLS ([[Part:BBa_K1119001|BBa_K1119001]]) was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format([[Part:BBa_K648013|BBa_K648013]]). The translation unit was driven by CMV promoter ([[Part:BBa_K1119006|BBa_K1119006]]) and terminated by hGH polyA signal ([[Part:BBa_K404108|BBa_K404108]]).
 
  
  

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