Team:Hong Kong HKUST/characterization/ef1a

Introduction

The constitutive Human Elongation Factor-1alpha (EF-1alpha) promoter regulates gene expression in mammalian cells. Up to now, only Pcmv has been used widely as a constitutive mammalian promoter in the iGEM competition. Here we introduce the EF-1alpha promoter that is known to be a consistently strong promoter in many cell types. The origin of this part is the Homo sapiens chromosome 6 genomic contig, GRCh37. p13.

For our characterization of this part, the DNA sequence of EF-1alpha Promoter was assembled with GFP reporter (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. Our negative control was the same as the experimental construct, but without the EF-1alpha promoter. EF-1alpha promoter efficiency was compared with that of the CMV promoter by transfecting GFP reporter driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108). Detailed protocols for our characterization work can be accessed via the link.

Method

We tried characterization of EF-1alpha promoter by obtaining in vivo expression of green fluorescence protein in mammalian cell. To do so, we have built an EF-1alpha characterization construct by fusing EF-1alpha promoter with green fluorescence protein (GFP) and hGH polyadenylation sequence (hGH pA) in pSB1C3 backbone. The construct was transfected in HEK293FT cells along with GFP – pSB1C3 construct for negative control and iDUET101a plasmid for positive control. We have cloned EF-1alpha promoter out from iDUET101a (Addgene), which contains EF-1alpha promoter and EGFP reporter. In addition, to compare promoter efficiency of EF-1alpha, we also transfected CMV promoter fused with green fluorescence and hGH polyadenylation sequence (hGH pA) in pSB1C3 backbone. The green fluorescence was observed under fluorescence microscope.

Characterization Procedure

1. Build:

- EF-1alpha characterization construct: EF-1alpha promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3 (BBa_K1119004 – BBa_K648013 – BBa_K404108 – pSB1C3)

- CMV comparison construct: CMV promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3 (BBa_K1119006 – BBa_K648013 – BBa_K404108 – pSB1C3)

- Negative control construct: GFP – hGH pA - pSB1C3 (BBa_K648013 – BBa_K404108 – pSB1C3)

2. Prepare iDUET101a (Addgene) in which EF-1alpha promoter was cloned from. This plasmid contains EF-1alpha promoter and EGFP reporter. We have transfected this plasmid for positive control for EF-1alpha characterization.

3. Culture HEK293FT cell line (see below)

4. Transfect EF-1alpha characterization construct, CMV construct, negative control and positive control plasmids in HEK293FT cell line.

5. Observe GFP signal under fluorescence microscope

Cell Culture and Transfection

We cultured HEK 293FT cells following ATCC’s standard procedure, except that we have used DMEM with 10% FBS and 1% penicillin/streptomycin for culture medium. For transfection, we have followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen). We have used serum free and antibiotics free DMEM to form DNA-lipofectamine complex.

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) .