Difference between revisions of "Part:BBa K1119011"
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This is a mammalian GFP generator used for characterization of our EF-1alpha promoter ([[Part:BBa_K1119010|BBa_K1119010]]). This GFP generator contains the EF-1alpha promoter sequence with GFP reporter in RFC25 format([[Part:BBa_K648013|BBa_K648013]]) and hGH polyA terminator ([[Part:BBa_K404108|BBa_K404108]]).
 
This is a mammalian GFP generator used for characterization of our EF-1alpha promoter ([[Part:BBa_K1119010|BBa_K1119010]]). This GFP generator contains the EF-1alpha 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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The EF-1alpha promoter-GFP was then transfected into HEK293FT cells and <i>in vivo</i> green fluorescence signal was observed under fluorescence microscope.  The positive control was iDUET101a plasmid (Addgene plasmid 17629) that contains EF-1alpha promoter and EGFP reporter. A negative control was made by GFP generator that does not contain the EF-1alpha promoter. EF-1alpha promoter efficiency was compared with that of CMV promoter by transfecting GFP reporter driven by CMV promoter ([[Part:BBa_K1119006|BBa_K1119006]]) and terminated by hGH polyA signal ([[Part:BBa_K404108|BBa_K404108]]).
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The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/ef1a detailed description] of our characterization can be found in HKUST iGEM 2013 Wiki.
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[[File:Final Final EF1A compiled.jpg|900px|thumb|center|'''Figure 1: GFP signal of EF-1alpha was observed.''' HEK293FT cells were transfected with iDUET101a (positive control), PEF-1alpha-GFP, PCMV-GFP (alternative mammalian constitutive promoter), and GFP without promoter. Cells transfected with PEF-1alpha-GFP showed green signal, similar to those with iDUET101a and PCMV-GFP. Our negative control, GFP without promoter did not give any GFP signal.]]
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<i>At the time of regional jamboree, no GFP signal of EF-1alpha could be observed. The sequence of EF-1alpha promoter cloned from iDUET101a contained full sequence of functional promoter region labeled in pBudCE4.1 (Invitrogen). We believed that EF-1alpha triggered transcription of GFP but failed to translate the GFP coding sequence due to short 5’ untranslated region. After regional jamboree, the promoter was re-cloned with additional junk sequences after promoter region to elongate 5’ untranslated region. This resulted in successful translation of GFP and green signal was observed.</i>
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<i><b>Reference</b></i>
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Qin, Jane Yuxia, Li Zhang, et al. "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter." PLoS ONE. 5.5 (2010) <http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010611>.
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Revision as of 12:01, 26 October 2013

Human Elongation Factor-1alpha Promoter - GFP - hGH polyA tail

This is a mammalian GFP generator used for characterization of our EF-1alpha promoter (BBa_K1119010). This GFP generator contains the EF-1alpha promoter sequence with GFP reporter in RFC25 format(BBa_K648013) and hGH polyA terminator (BBa_K404108).

The EF-1alpha promoter-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under fluorescence microscope. The positive control was iDUET101a plasmid (Addgene plasmid 17629) that contains EF-1alpha promoter and EGFP reporter. A negative control was made by GFP generator that does not contain the EF-1alpha promoter. EF-1alpha promoter efficiency was compared with that of CMV promoter by transfecting GFP reporter driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108).

The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/ef1a detailed description] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1: GFP signal of EF-1alpha was observed. HEK293FT cells were transfected with iDUET101a (positive control), PEF-1alpha-GFP, PCMV-GFP (alternative mammalian constitutive promoter), and GFP without promoter. Cells transfected with PEF-1alpha-GFP showed green signal, similar to those with iDUET101a and PCMV-GFP. Our negative control, GFP without promoter did not give any GFP signal.

At the time of regional jamboree, no GFP signal of EF-1alpha could be observed. The sequence of EF-1alpha promoter cloned from iDUET101a contained full sequence of functional promoter region labeled in pBudCE4.1 (Invitrogen). We believed that EF-1alpha triggered transcription of GFP but failed to translate the GFP coding sequence due to short 5’ untranslated region. After regional jamboree, the promoter was re-cloned with additional junk sequences after promoter region to elongate 5’ untranslated region. This resulted in successful translation of GFP and green signal was observed.

Reference

Qin, Jane Yuxia, Li Zhang, et al. "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter." PLoS ONE. 5.5 (2010) <http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010611>.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 326
    Illegal AgeI site found at 83
    Illegal AgeI site found at 1067
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1470
    Illegal BsaI.rc site found at 972