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.

Characterization Procedure

1. Build:

- EF-1alpha characterization construct: EF-1alpha promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3 (BBa_K1119010 – 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 American Type Culture Collection’s standard procedure, except that we used DMEM with 10% FBS and 1% penicillin/streptomycin in our culture medium. For transfection, we followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen) and used serum-free and antibiotics-free DMEM to form the DNA-lipofectamine complex.

Result

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. Scale bar = 0.1mm

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.

Conclusion

The sequence of EF-1alpha promoter cloned from iDUET101a contains the 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 CDS due to having a too short 5’ untranslated region. Additional junk sequences should be added before the first start codon to elongate the 5’ untranslated region, which may lead to more 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) .