Team:Hong Kong HKUST/characterization/ef1a

Introduction

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.

In our characterization, the coding sequence of EF-1alpha Promoter was assembled with GFP reporter (BBa_BBa_K648013) and hGH polyA terminator (BBa_K404108) using Freiburg’s RFC25 format. The EF1alpha 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 detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.

Result

Figure 1: No GFP signal of EF-1alpha promoter was observed. While cells transfected with iDUET and pCMV-GFP showed GFP signal, those transfected with EF-1alpha promoter-GFP did not gave GFP signals. Our negative control, GFP without promoter did not gave any GFP signals. Scale bar = 10 microns

Conclusion

The sequence of EF-1alpha promoter cloned from iDUET101a contains full sequence of functional promoter region labeled in pBudCE4.1 (Invitrogen). We believe that EF-1alpha triggers transcription of GFP but fails to translate the GFP coding sequence due to short 5’ untranslated region. Additional junk sequences should be added before the first start codon to elongate 5’ untranslated region for successful translation.

Reference

Qin, Jane Yuxia, Li Zhang, et al. "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter." PLoS ONE. 5.5 (2010) .