Team:Hong Kong HKUST/characterization/ef1a

From 2013.igem.org

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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>In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format(BBa_K648013). The translation unit was driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108).
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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 MLS-GFP generator (BBa_K1119009) was then transfected into HEK293FT cells. Mitochondria were stained after transfection and co-localization was determined by area of signal that overlapped.
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To provide a positive control, CDS of EGFP from pEGFP-N1 (Clontech) was inserted downstream and in frame with the CDS of the MLS in the commercial plasmid pCMV/myc/mito, (Invitrogen, Carlsbard, CA). A negative control was made by GFP generator that does not contains the CDS of MLS (BBa_K1119008).
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The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.</p>
The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.</p>
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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><center><img src="https://static.igem.org/mediawiki/parts/a/ab/Final_Final_EF1A_compiled.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: 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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<img src="https://static.igem.org/mediawiki/parts/8/80/Scatterplots_mlsquantification.jpg" style="width:700px;height:268px;">
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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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<img src="https://static.igem.org/mediawiki/parts/c/c2/Barchart_mlsquantification.jpg" >
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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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<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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<div class="nine columns"><p id="reference"></p>
<div class="nine columns"><p id="reference"></p>
<h3>Reference</h3>
<h3>Reference</h3>
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<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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Revision as of 15:36, 27 September 2013

EF1-alpha Promoter

Introduction

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). The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.


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) .

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