Team:Hong Kong HKUST/characterization/cmv

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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=#cp>Characterization Procedure</a>
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<a href="">Mitochondrial Leader Sequence</a>
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<a href="">CMV Promoter</a>
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<a href="">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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<div class="nine columns"><p id="introduction"></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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<h3>Introduction</h3>
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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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<p>CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter. </p>
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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 detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.</p>
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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 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. 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 protocols</a> employed for our characterization work can be accessed via the link.</p>
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<h3>Characterization Procedure</h3>
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1. Build:<p>
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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)</p>
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<p>-      Negative control construct: GFP – hGH pA - pSB1C3
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(BBa_K648013 – BBa_K404108 – pSB1C3)</p>
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<p>2.    Culture HEK293FT cell line (see below)</p>
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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.</p>
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<p>4.    Observe GFP signal under confocal microscope
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<h3>Cell Culture and Transfection</h3>
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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><img src="https://static.igem.org/mediawiki/parts/b/b9/Mlschar_1.jpg" >
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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>
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<br><p><b>Figure 1. MLS directs GFP into mitochondria.</b> When MLS is added to the N terminus of GFP, the GFP was directed to the mitochondria in the cells, giving patches of GFP signal that overlapped with the signals from MitoTracker®. When MLS is not added to the GFP, the GFP signal can be seen scattered all around in the cell. 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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<br><p><b>Figure 2. Scatter plots of fluorescence intensities of green (y axis) and red (x axis) from images shown in Figure 1</b>. It showed that the BioBrick MLS-GFP and commercial GFP construct had linear relationship of green intensities and red intensities while the GFP generator had no relationship. Pearson's correlation coefficient (rp) and Spearman correlation coefficient (rs) were determined using the Pearson-Spearman correlation colocalization plugin (French et al., 2008) for ImageJ with a threshold of 0 and listed for each image.</p>
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<br><p><b>Figure 3. Mean Pearson correlation coefficient(rp) and mean Spearman correlation coefficient(rs) were shown in bar chart.</b> Using ImageJ software and plugins, the Pearson correlation coefficient and Spearman correlation coefficient were generated. For every batch of transfected cells, four samples were used for quantification. Experimental BioBrick MLS-GFP and commercial MLS-GFP: Coefficients were close to 1, good colocalization ; GFP: Coefficients were close to 0, poor colocalization. Error bars show standard deviation.</p>
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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>
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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

Retrieved from "http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv"