Difference between revisions of "Team:OUC-China/Results"

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{{OUC-China}}
 
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<p>Here you can describe the results of your project and your future plans. </p>
 
  
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<h5>What should this page contain?</h5>
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<ul>
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<script type="text/javascript">
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<li> Clearly and objectively describe the results of your work.</li>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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</script>
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<ul>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<h5>Inspiration</h5>
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<p>See how other teams presented their results.</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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<li class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">T</span>est previous stem-loops</a></li>
 +
<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">M</span>easure a series of designed stem-loops</a></li>
 +
<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">C</span>onstruct a wonderful toolkit</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">F</span>urther exploration of free energy</a></li>
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<br id="float01">
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<br>
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<h3 class="text-center">TEST PREVIOUS STEM-LOOPS</h3>
 +
<br>
 +
<p>It is reported that there are several native stem-loops that have effects on its flanking genes, either at the 3’ end or the 5’ end[1]. Ergo, we use two native stem-loops from <i>R. capsulatus</i> and <i>E.coli</i>[2] and a previously experimented stem-loops with different free energy to preliminary verify that stem-loops in the intergenic region can regulate the relative expression of two reporter genes within polycistrons. We measured them on both transcriptional and translational level.</p>
 +
<p>The result see as follows:</p>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/5/5e/T--OUC-China--result1.png" class="img-responsive" alt="Folding -25.6">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;">Figure.1  Both relative expression on RNA and protein level with stem-loop of -25.6 kcal/mol (measured by Mfold) are significantly higher than those of control group without stem-loop, which means stem-loops can coordinate the expression within cistrons. The vertical coordinate is the ratio of upstream gene gfp to downstream gene mcherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
 +
<p>After the function and sequences of stem-loops confirmed, we standardized and submitted them to iGEM part registry with detailed description.</p>
 +
<p><a href="https://2016.igem.org/Team:OUC-China/Part_Collection">Click here</a> to see more.</p>
 +
<p>Results suggested that stem-loops at 3’ end functioned well in regulation. Hence we tested another two native stem-loops in the following.</p>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/7/73/T--OUC-China--pr-3-4.jpg" class="img-responsive" alt="stem-loops-123">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;" class="text-center">Figure.2 Structures of three previous stem-loops</p>
 +
<div class="clearfix"></div>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/9/95/T--OUC-China--result3.jpg" class="img-responsive" alt="toolkit">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;">Figure.3 Stem-loops with lower folding free energy lead to stronger protection of upstream gene, which results in higher ratio of GFP to mCherry both in transcriptional and translational level. Error bars indicate s.d. of mean of experiments in triplicate</p>
 +
<br id="float02">
 +
<hr>
 +
<h3 class="text-center">MEASURE A SERIES OF DESIGNED STEM-LOOPS</h3>
 +
<br>
 +
<p>In addition to the confirmation of previous stem-loops, we also devoted to exploring new stem-loops designed by ourselves. It is difficult to estimate the relationship between structure and energy, thus we weighed several software and finally chose Mfold, a commonly acknowledged one. Employing the same rules, we designed several stem-loops (Figure.4) with various free folding energy and tested them (Figure.5). As shows, these parts tune the ratio of the upstream and downstream genes as well.</p>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/d/d5/T--OUC-China--pr-3-6.jpg" class="img-responsive" alt="1">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;" class="text-center">Figure.4 Structure of three stem-loops designed by ourselves</p>
 +
<div class="clearfix"></div>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/e/e9/T--OUC-China--result5.png" class="img-responsive" alt="2">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;">Figure.5 Both relative expression on RNA and protein level of designed stem-loops of -30.1, -34.4,38.8,44.9kcal/mol (measured by Mfold) compare to the control group without stem-loop. The vertical coordinates is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
 +
<br id="float03">
 +
<hr>
 +
<h3 class="text-center">CONSTRUCT A WONDERFUL TOOLKIT</h3>
 +
<br>
 +
<p>Collecting those validated previous stem-loops and our designed ones, we got a set of stem-loops to form a toolkit. We measured them both in transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. With the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library.</p>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/3/30/T--OUC-China--proof-444.png" class="img-responsive" alt="1">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;" class="text-center">Figure.6 Structure of three stem-loops designed by ourselves</p>
 +
<br id="float04">
 +
<hr>
 +
<h3 class="text-center">FURTHER EXPLORATION OF FREE ENERGY</h3>
 +
<br>
 +
<p>We measured them both on transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. And with the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library. Below is the relative expression on two levels of different stem-loops in our toolkit.</p>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/7/79/T--OUC-China--proof-555.png" class="img-responsive" alt="1">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;" class="text-center">Figure.7 RNA varying with folding free energy of stem-loops</p>
 +
<div class="clearfix"></div>
 +
<div class="row">
 +
<div class="col-md-2"></div>
 +
<div class="col-md-8">
 +
<img src="https://static.igem.org/mediawiki/2016/b/b7/T--OUC-China--result7.png" class="img-responsive" alt="2">
 +
</div>
 +
<div class="col-md-2"></div>
 +
</div>
 +
<p style="font-size:16px;" class="text-center">Figure.8 Protein expression varying with folding free energy of stem-loop</p>
 +
<p>There may be certain relationship between the relative expression and free energy, so we did more to explore the relationship, see details at <a href="https://2016.igem.org/Team:OUC-China/Model">model</a>.</p>
 +
</div>
 +
</div>
 +
<div class="oucBanner">
 +
        <h3 class="text-center">Cistrons Concerto</h3>
 +
</div>
 +
<div class="row oucInformation">
 +
<div class="col-md-1"></div>
 +
<div class="col-md-5">
 +
<h3>Thanks</h3>
 +
<p>1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences</p>
 +
<p>2.NEW ENGLAND Biolabs</p>
 +
<p>3.GenScript</p>
 +
</div>
 +
<div class="col-md-5 Contact">
 +
<h3>Contact us:</h3>
 +
<p><b>E-mail</b>: oucigem@163.com</p>
 +
<p><b>Designed and built</b> by @ Jasmine Chen and @ Zexin Jiao</p>
 +
<p>We are OUC-iGEM
 +
<img src="https://static.igem.org/mediawiki/2016/5/58/T--OUC-China--foot4.png" width="70" height="70" alt="logo-one">
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 +
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<div class="oucBottom">
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</body>
 
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Latest revision as of 03:47, 23 November 2016

Results

project-banner


TEST PREVIOUS STEM-LOOPS


It is reported that there are several native stem-loops that have effects on its flanking genes, either at the 3’ end or the 5’ end[1]. Ergo, we use two native stem-loops from R. capsulatus and E.coli[2] and a previously experimented stem-loops with different free energy to preliminary verify that stem-loops in the intergenic region can regulate the relative expression of two reporter genes within polycistrons. We measured them on both transcriptional and translational level.

The result see as follows:

Folding -25.6

Figure.1 Both relative expression on RNA and protein level with stem-loop of -25.6 kcal/mol (measured by Mfold) are significantly higher than those of control group without stem-loop, which means stem-loops can coordinate the expression within cistrons. The vertical coordinate is the ratio of upstream gene gfp to downstream gene mcherry. Error bars indicate s.d. of mean of experiments in triplicate.

After the function and sequences of stem-loops confirmed, we standardized and submitted them to iGEM part registry with detailed description.

Click here to see more.

Results suggested that stem-loops at 3’ end functioned well in regulation. Hence we tested another two native stem-loops in the following.

stem-loops-123

Figure.2 Structures of three previous stem-loops

toolkit

Figure.3 Stem-loops with lower folding free energy lead to stronger protection of upstream gene, which results in higher ratio of GFP to mCherry both in transcriptional and translational level. Error bars indicate s.d. of mean of experiments in triplicate


MEASURE A SERIES OF DESIGNED STEM-LOOPS


In addition to the confirmation of previous stem-loops, we also devoted to exploring new stem-loops designed by ourselves. It is difficult to estimate the relationship between structure and energy, thus we weighed several software and finally chose Mfold, a commonly acknowledged one. Employing the same rules, we designed several stem-loops (Figure.4) with various free folding energy and tested them (Figure.5). As shows, these parts tune the ratio of the upstream and downstream genes as well.

1

Figure.4 Structure of three stem-loops designed by ourselves

2

Figure.5 Both relative expression on RNA and protein level of designed stem-loops of -30.1, -34.4,38.8,44.9kcal/mol (measured by Mfold) compare to the control group without stem-loop. The vertical coordinates is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.


CONSTRUCT A WONDERFUL TOOLKIT


Collecting those validated previous stem-loops and our designed ones, we got a set of stem-loops to form a toolkit. We measured them both in transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. With the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library.

1

Figure.6 Structure of three stem-loops designed by ourselves


FURTHER EXPLORATION OF FREE ENERGY


We measured them both on transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. And with the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library. Below is the relative expression on two levels of different stem-loops in our toolkit.

1

Figure.7 RNA varying with folding free energy of stem-loops

2

Figure.8 Protein expression varying with folding free energy of stem-loop

There may be certain relationship between the relative expression and free energy, so we did more to explore the relationship, see details at model.

Cistrons Concerto

Thanks

1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

2.NEW ENGLAND Biolabs

3.GenScript

Contact us:

E-mail: oucigem@163.com

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