Team:Hong Kong HKUST/characterization/cmv

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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<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>
 
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
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<h6>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a>
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<h6>Mitochondrial Leader Sequence</h6>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/mls">Mitochondrial Leader Sequence</a>
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CMV Promoter<ul><li>
<a href=#introduction>Introduction</a>
<a href=#introduction>Introduction</a>
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<a href=#method>Method</a>
 
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<a href=#reference>Reference</a>
<a href=#reference>Reference</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</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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<h6>CMV Promoter</h6>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/ef1a">EF1-alpha Promoter</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization/ef1a">EF-1alpha Promoter</a>
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<p>CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter. </p>
<p>CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter. </p>
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<p>In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108).
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<p>For this promoter's characterization we assembled it with 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>).
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The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.
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The P<i><sub>cmv</sub></i>-GFP was then transfected into HEK293FT cells and <i>in vivo</i> green fluorescence signal was observed under confocal microscope.
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The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator (BBa_K648013) that does not contain the CMV promoter.
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The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. The negative control was the same as the experimental construct, but minus the promoter.
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The <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">detailed protocol</a> of our characterization can be found in HKUST iGEM 2013 Wiki.</p>
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The <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">detailed protocols</a> employed for our characterization work can be accessed via the link.</p>
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<h3>Method</h3>
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CMV promoter was characterized by obtaining in vivo expression of green fluorescence protein in mammalian cell. To do so, we have built a CMV characterization construct by fusing CMV 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 pEGFP-N1 for positive control. We have cloned CMV promoter out from pEGFP-N1 (Addgene), which contains CMV promoter and EGFP reporter. The green fluorescence was observed under confocal microscope.
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<h3>Characterization Procedure</h3>
<h3>Characterization Procedure</h3>
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1. Build:
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1. Build:<p>
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-      CMV characterization construct: CMV promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3
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<p>-      CMV promoter characterization 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.    Culture HEK293FT cell line (see below)
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<p>2.    Culture HEK293FT cell line (see below)</p>
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3.    Transfect CMV characterization (CMV – GFP – hGH pA – pSB1C3), negative control (GFP – hGH pA – pSB1C3) and positive control (pEGFP-N1) plasmids in HEK293FT cell line.
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<p>3.    Transfect CMV promoter characterization (CMV – GFP – hGH pA – pSB1C3), negative control (GFP – hGH pA – pSB1C3) and positive control (pEGFP-N1) plasmids in HEK293FT cell line.
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*We cloned CMV promoter out from pEGFP-N1 (Addgene) that contains CMV promoter and enhanced green fluorescence protein (EGFP). We used this plasmid as our positive control for CMV promoter characterization.
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*We cloned CMV promoter out from pEGFP-N1 (Addgene) that contains CMV promoter and enhanced green fluorescence protein (EGFP). We used this plasmid as our positive control for CMV promoter characterization.</p>
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4.    Observe GFP signal under confocal microscope
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<p>4.    Observe GFP signal under confocal microscope
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<h3>Cell Culture and Transfection</h3>
<h3>Cell Culture and Transfection</h3>
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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 HEK293FT 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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<h3>Result</h3><br><center><img src="https://static.igem.org/mediawiki/parts/thumb/c/c6/Final_CMV_annotated_no_ABC.jpg/600px-Final_CMV_annotated_no_ABC.jpg" ></center>
<h3>Result</h3><br><center><img src="https://static.igem.org/mediawiki/parts/thumb/c/c6/Final_CMV_annotated_no_ABC.jpg/600px-Final_CMV_annotated_no_ABC.jpg" ></center>
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<br><p><b>Figure 1. CMV promoter drives expression of GFP</b>. HEK cells transfected with pCMV-GFP gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10 microns</p>
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<br><p><b>Figure 1. CMV promoter drives expression of GFP.</b> HEK293FT cells transfected with P<i><sub>cmv</sub></i>-GFP gave GFP signals. HEK293FT cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10µm.</p>
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<h3>Conclusion</h3>
<h3>Conclusion</h3>
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CMV promoter was observed to drive expression of GFP in HEK293FT cells and green fluorescence signal was obtained under confocal microscope.
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<h3>Reference</h3>
<h3>Reference</h3>
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BD Biosciences Clontech. (2002). pEGFP-N1 Vector Information. Retrieved from http://www.staff.ncl.ac.uk/p.dean/pEGFP-N1_map.pdf
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Latest revision as of 13:47, 28 October 2013

CMV Promoter

Introduction

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.


For this promoter's characterization we assembled it with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108). The Pcmv-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope. The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. The negative control was the same as the experimental construct, but minus the promoter. The detailed protocols employed for our characterization work can be accessed via the link.

Characterization Procedure

1. Build:

- CMV promoter characterization 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. Culture HEK293FT cell line (see below)

3. Transfect CMV promoter characterization (CMV – GFP – hGH pA – pSB1C3), negative control (GFP – hGH pA – pSB1C3) and positive control (pEGFP-N1) plasmids in HEK293FT cell line. *We cloned CMV promoter out from pEGFP-N1 (Addgene) that contains CMV promoter and enhanced green fluorescence protein (EGFP). We used this plasmid as our positive control for CMV promoter characterization.

4. Observe GFP signal under confocal microscope

Cell Culture and Transfection

We cultured HEK293FT 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. CMV promoter drives expression of GFP. HEK293FT cells transfected with Pcmv-GFP gave GFP signals. HEK293FT cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10µm.


Conclusion

CMV promoter was observed to drive expression of GFP in HEK293FT cells and green fluorescence signal was obtained under confocal microscope.

Reference

BD Biosciences Clontech. (2002). pEGFP-N1 Vector Information. Retrieved from http://www.staff.ncl.ac.uk/p.dean/pEGFP-N1_map.pdf