Regulatory

Part:BBa_K1119010

Designed by: Wai In Hui   Group: iGEM13_Hong_Kong_HKUST   (2013-10-24)
Revision as of 12:19, 26 October 2013 by Hjleeab (Talk | contribs)

Human Elongation Factor-1alpha Promoter

The constitutive Human Elongation Factor-1alpha (EF-1alpha) Promoter regulates gene expression in mammalian cells. While only CMV is widely used for a constitutive mammalian promoter in iGEM, here we introduce EF-1alpha promoter that is known to be a consistent strong promoter in many cell types. The origin of this part is Homo sapiens chromosome 6 genomic contig, GRCh37. p13.

Characterization

In our characterization, the coding sequence of EF-1alpha Promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108) using Freiburg’s RFC25 format. 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 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 AgeI site found at 83
  • 1000
    COMPATIBLE WITH RFC[1000]


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