Composite

Part:BBa_K1119011

Designed by: Wai In Hui   Group: iGEM13_Hong_Kong_HKUST   (2013-10-24)

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 driving a 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 Number 17629) that contains EGFP reporter driven by an EF-1alpha promoter. A negative control was made by GFP generator that does not contain the EF-1alpha promoter. As a side by side comparison, a CMV promoter driven GFP reporter was also transfected, though a quantitative comparison between the two was not conducted in our characterization.

The 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 weaker green signal compared to those with iDUET101a and pCMV-GFP. This result agreed with the result reported by Qin et al., that the EF-1alpha promoter gives weaker activity than CMV promoter in HEK293T cells. Our negative control, GFP without promoter did not give any GFP signal. Scale bar = 0.1mm

At the time of regional jamboree, no GFP signal could be observed in cells transfected with GFP reporter driven by EF-1alpha promoter. Originally, we thought that the sequence of EF-1alpha promoter cloned from iDUET101a contained the full functional promoter region annotated in pBudCE4.1 (Invitrogen). We believed that EF-1alpha did trigger transcription but failed to translate the GFP coding sequence due to insufficient 5’ untranslated region (UTR). After the regional jamboree, the promoter was re-cloned with additional 3' sequences after the identified TATA box to allow a longer 5’ untranslated region before the GFP coding DNA sequence. From the the results above, we believed that translation of GFP is successful this time.

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>.

Zhou, B. Y., Ye, Z., Chen, G., Gao, Z. P., Zhang, Y. A., & Cheng, L. (2007). Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. Stem Cells, 25(3), 779-789. doi:10.1634/stemcells.2006-0128 <http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2006-0128/abstract>


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


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