Team:Hong Kong HKUST/characterization/ef1a

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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/experiment">Experiments</a></li>
 
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<h6>Mitochondrial Leader Sequence</h6>
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<a href=#introduction>Introduction</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/mls">Mitochondrial Leader Sequence</a>
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<a href=#result>Result</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv">CMV Promoter</a>
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<a href=#conclusion>Conclusion</a>
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EF-1alpha Promoter<ul><li>
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<a href=#introduction>Introduction</a>
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<a href=#reference>Reference</a>
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<a href=#method>Method</a>
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<a href=#cp>Characterization Procedure</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization>Characterization</a>
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<a href=#ct>Cell Culture and Transfection</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv">CMV Promoter</a>
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<a href=#result>Result</a>
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<h6>EF1-alpha Promoter</h6>
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<a href=#conclusion>Conclusion</a>
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<h2 class="centered">EF1-alpha Promoter</h2>
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<h2 class="centered">EF-1alpha Promoter</h2>
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<div class="nine columns"><p id="introduction"></p>
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<h3>Introduction</h3>
<h3>Introduction</h3>
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<p>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.</p>
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<p>The constitutive human Elongation Factor-1alpha (EF-1alpha) Promoter regulates gene expression in mammalian cells. It is known that the CMV promoter is commonly used for constitutive expression, and here we introduce EF-1alpha promoter as an alternative mammalian promoter, which works in a wide range of cell types. The origin of this part is from <i>Homo sapiens</i> chromosome 6 genomic contig, GRCh37. p13.</p>
<br>
<br>
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<p>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).
 
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The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.</p>
 
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<p>In our characterization, the sequence of EF-1alpha Promoter was assembled in front of a GFP reporter (<a href="http://parts.igem.org/Part:BBa_K648013">BBa_K648013</a>) and hGH polyA terminator (<a href="http://parts.igem.org/Part:BBa_K404108">BBa_K404108</a>) 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 (<a href="http://www.addgene.org/17629/">Addgene Plasmid Number 17629</a>) 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.
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<h3>Method</h3>
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<p>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Detailed protocols</a> for our characterization work can be accessed via the link.</p>
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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.
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<div class="nine columns"><p id="cp"></p>
<h3>Characterization Procedure</h3>
<h3>Characterization Procedure</h3>
<p>
<p>
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1.    Build:
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<p>1.    Build:</p>
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-      EF-1alpha characterization construct: EF-1alpha promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3
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<p>-      EF-1alpha characterization construct: EF-1alpha promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3
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(BBa_K1119004 – BBa_K648013 – BBa_K404108 – pSB1C3)
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(BBa_K1119010 – BBa_K648013 – BBa_K404108 – pSB1C3)</p>
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-      CMV comparison construct: CMV promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3
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<p>-      CMV comparison construct: CMV promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3
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(BBa_K1119006 – BBa_K648013 – BBa_K404108 – pSB1C3)
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(BBa_K1119006 – BBa_K648013 – BBa_K404108 – pSB1C3)</p>
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-      Negative control construct: GFP – hGH pA - pSB1C3
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<p>-      Negative control construct: GFP – hGH pA - pSB1C3
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(BBa_K648013 – BBa_K404108 – pSB1C3)
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(BBa_K648013 – BBa_K404108 – pSB1C3)</p>
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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.
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<p>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.</p>
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3.    Culture HEK293FT cell line (see below)
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<p>3.    Culture HEK293FT cell line (see below)</p>
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4.    Transfect EF-1alpha characterization construct, CMV construct, negative control and positive control plasmids in HEK293FT cell line.
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<p>4.    Transfect EF-1alpha characterization construct, CMV construct, negative control and positive control plasmids in HEK293FT cell line.</p>
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5.    Observe GFP signal under fluorescence microscope
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<p>5.    Observe GFP signal under fluorescence microscope
</p>
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<h3>Cell culture and transfection</h3>
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<h3>Cell Culture and Transfection</h3>
<p>
<p>
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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.  
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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.  
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<div class="nine columns"><p id="result"></p>
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<h3>Result</h3><img src="https://static.igem.org/mediawiki/parts/a/ab/Final_Final_EF1A_compiled.jpg" style="padding-left:5px;width:90%;padding-top:5px;">
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<h3>Result</h3><img src="https://static.igem.org/mediawiki/parts/0/06/Final_Final_EF1A_compiled.png" style="padding-left:5px;width:90%;padding-top:5px;">
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<br><p><b>Figure 1: No GFP signal of EF-1alpha promoter was observed. </b>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</p>
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<br><p><b>Figure 1: GFP signal of EF-1alpha observed.</b> 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</p>
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<br><p>
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<i>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.</i>
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<h3>Conclusion</h3>
<h3>Conclusion</h3>
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<p>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.</p>
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<p>The EF-1alpha promoter is fully functional and has a weaker transcriptional strength than the CMV promoter in HEK293FT cells.</p>
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<h3>Reference</h3>
<h3>Reference</h3>
<p>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>.</p>
<p>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>.</p>
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<p>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></p>
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Latest revision as of 03:41, 29 October 2013

EF-1alpha Promoter

Introduction

The constitutive human Elongation Factor-1alpha (EF-1alpha) Promoter regulates gene expression in mammalian cells. It is known that the CMV promoter is commonly used for constitutive expression, and here we introduce EF-1alpha promoter as an alternative mammalian promoter, which works in a wide range of cell types. The origin of this part is from Homo sapiens chromosome 6 genomic contig, GRCh37. p13.


In our characterization, the sequence of EF-1alpha Promoter was assembled in front of a 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 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. 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 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.



Conclusion

The EF-1alpha promoter is fully functional and has a weaker transcriptional strength than the CMV promoter in HEK293FT cells.

Reference

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

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