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

Line 66: Line 66:
 
  f3 = $("#float03").offset().top;
 
  f3 = $("#float03").offset().top;
 
  f4 = $("#float04").offset().top;
 
  f4 = $("#float04").offset().top;
−
 
+
  f3 = $("#float05").offset().top;
 +
  f4 = $("#float06").offset().top;
  
 
 
 
 
Line 87: Line 88:
 
  fixRight.eq(3).addClass('active').siblings().removeClass('active');
 
  fixRight.eq(3).addClass('active').siblings().removeClass('active');
 
  }    
 
  }    
−
 
+
  if(sTop>=f5-100){
 +
  fixRight.eq(4).addClass('active').siblings().removeClass('active');
 +
  }
 +
  if(sTop>=f6-100){
 +
  fixRight.eq(5).addClass('active').siblings().removeClass('active');
 +
  }
 
 
 
    }
 
    }
Line 165: Line 171:
 
<div class="col-md-3" id="myScrollspy">
 
<div class="col-md-3" id="myScrollspy">
 
<ul class="" data-spy="affix" data-offset-top="225" data-offset-bottom="150" id="myNav">
 
<ul class="" data-spy="affix" data-offset-top="225" data-offset-bottom="150" id="myNav">
−
<li class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">T</span>est previous stem-loops</a></li>
+
<li class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">N</span>ative stem-loops</a></li>
−
<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">D</span>esign our stem-loops</a></li>
+
<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">D</span>esigned stem-loops</a></li>
−
<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">S</span>tem-loops Designed by Ourselves</a></li>
+
<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">S</span>tandardize parts</a></li>
−
<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">O</span>ur Toolkit</a></li>
+
<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">C</span>onstruct toolkit</a></li>
 +
<li><a href="#float05"><span style="font-family:'Lucida Calligraphy';font-size:22px;">S</span>oftware</a></li>
 +
<li><a href="#float06"><span style="font-family:'Lucida Calligraphy';font-size:22px;">M</span>odel</a></li>
 
<a id="BtnTop">
 
<a id="BtnTop">
 
<img src="https://static.igem.org/mediawiki/2016/d/d2/T--OUC-China--btt.png" alt="Back to top">
 
<img src="https://static.igem.org/mediawiki/2016/d/d2/T--OUC-China--btt.png" alt="Back to top">
Line 175: Line 183:
 
</div>
 
</div>
 
<div class="col-md-9 hpMain">
 
<div class="col-md-9 hpMain">
−
<div class="row">
 
−
<div class="col-md-2"></div>
 
−
<div class="col-md-8">
 
−
<img src="https://static.igem.org/mediawiki/2016/a/a0/T--OUC-China--pr-3-1.jpg" class="img-responsive" alt="">
 
−
</div>
 
−
<div class="col-md-2"></div>
 
−
</div>
 
−
<p style="font-size:22px">This year, we devoted to the exploitation of new regulatory parts and the exploration of a kind of novel regulatory method.</p>
 
 
<br id="float01">
 
<br id="float01">
−
<br />
+
<br>
−
<h3 class="text-center">TEST PREVIOUS STEM-LOOPS</h3>
+
<h3 class="text-center">NATIVE STEM-LOOPS</h3>
−
<br />
+
<p>I. Validate that <b>native</b> stem-loops can affect the expression of its flanking cistron based on our platform <a href="https://2016.igem.org/Team:OUC-China/Results#float01">Click here</a> to know more.</p>
−
<p>To collect parts and modify as much new parts as possible for broad use, we began with standardization of the existing elements. Several native stem-loops had been discovered to have influence on flank genes expression and had been tested experimentally. However, they were only tested roughly, without quantitatively characterization of stem-loops. We further measured the stem-loops with our dual-fluorescent reporter system (Figure 1), and standardized them submitting to iGEM part registry with detailed description. Click here to see our <a href="https://2016.igem.org/Team:OUC-China/Parts">registried parts</a>.</p>
+
 
<div class="row">
 
<div class="row">
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
<div class="col-md-8">
 
<div class="col-md-8">
−
<img src="https://static.igem.org/mediawiki/2016/e/ee/T--OUC-China--pr-3-2.png" class="img-responsive" alt="Dual-fluorescent reporter system">
+
<img src="https://static.igem.org/mediawiki/2016/5/5e/T--OUC-China--proof-111.png" class="img-responsive" alt="native stem-loops">
 
</div>
 
</div>
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
</div>
 
</div>
−
<p class="text-center" style="font-size:16px;">Figure 1 Dual-fluorescent reporter system</p>
+
<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>We constructed the dual-fluorescent reporter system to test the regulatory effect of various stem-loops. After inserting stem-loops into the dual-fluorescent reporter system, we measured the relative expression of the upstream gfp and downstream mCherry to test regulatory effect on both transcriptional and translational level. The result we measured the stem-loop of -25.6kcal/mol is as follows:</p>
+
<p>II. Prove that native stem-loops with gradient folding free energy results in differential expression. <a href="https://2016.igem.org/Team:OUC-China/Results#float01">Click here</a> to know more.</p>
 
<div class="row">
 
<div class="row">
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
<div class="col-md-8">
 
<div class="col-md-8">
−
<img src="https://static.igem.org/mediawiki/2016/7/79/T--OUC-China--pr-3-3.png" class="img-responsive" alt="Dual-fluorescent reporter system">
+
<img src="https://static.igem.org/mediawiki/2016/a/a4/T--OUC-China--proof-222.jpg" class="img-responsive" alt="native stem-loops">
 
</div>
 
</div>
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
</div>
 
</div>
−
<p style="font-size:16px;">Figure 2  Relative expression on RNA and protein level with stem-loop of -25.6 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
+
<p style="font-size:16px;">Figure 2  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>
−
<p>Our result shows that the insert of stem-loop may have regulatory effects. Then we did more to explore. We tested another two stem-loops that have been used before and analyzed the results. The tested stem-loops and results are as follows.</p>
+
−
<div class="row">
+
−
<div class="col-md-2"></div>
+
−
<div class="col-md-8">
+
−
<img src="https://static.igem.org/mediawiki/2016/7/73/T--OUC-China--pr-3-4.jpg" class="img-responsive" alt="Structures of three discovered stem-loops">
+
−
</div>
+
−
<div class="col-md-2"></div>
+
−
</div>
+
−
<p class="text-center" style="font-size:16px;">Figure 3 Structures of three discovered stem-loops</p>
+
−
<div class="row">
+
−
<div class="col-md-2"></div>
+
−
<div class="col-md-8">
+
−
<img src="https://static.igem.org/mediawiki/2016/3/31/T--OUC-China--pr-3-5.jpg" class="img-responsive" alt="Structures of three discovered stem-loops">
+
−
</div>
+
−
<div class="col-md-2"></div>
+
−
</div>
+
−
<p style="font-size:16px;">Figure 4 Relative expression of 3 stem-loops with different folding free energy. Error bars indicate s.d. of mean of experiments in triplicate.</p>
+
−
<p>After made sure that the stem loops did have effects on regualtion, Moreover, we standardized the 3 elements and submit them to iGEM parts registry for others’ use.</p>
+
 
<br id="float02">
 
<br id="float02">
 
<hr>
 
<hr>
−
<h3 class="text-center" style="font-size:22px">DESIGN OUR STEM-LOOPS</h3>
+
<h3 class="text-center">DESIGNED STEM-LOOPS</h3>
−
<br />
+
<p>III. Verify the regulatory effect of designed stem-loops.</p>
−
<p>Besides the confirmation of previous discovered stem-loops, we also devoted to exploring new stem-loops designed by ourselves. The relationship between structure and nergy is a tricky problem and we weighed several software and finally we chose Mfold. Below are the result of testing of new designed stem-loops. The new designed stem-loops are as follows:</p>
+
 
<div class="row">
 
<div class="row">
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
<div class="col-md-8">
 
<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="Structures of three discovered stem-loops">
+
<img src="https://static.igem.org/mediawiki/2016/0/07/T--OUC-China--proof-333.jpg" class="img-responsive" alt="designed stem-loops">
 
</div>
 
</div>
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
</div>
 
</div>
−
<p class="text-center" style="font-size:16px;">Figure 5 Structure of three stem-loops designed by ourselves</p>
+
<p style="font-size:16px;">Figure 3 One of the designed stem-loop of -34.4 kcal/mol (measured by Mfold) inserted into intergenic regions can also tune the expression of gfp and mcherry as expected, similarly with the effect of native stem-loops. Error bars indicate s.d. of mean of experiments in triplicate.</p>
 +
<br id="float03">
 +
<hr>
 +
<h3 class="text-center">STANDARDIZE PARTS</h3>
 +
<p>IV. Standardize measured regulatory elements and submit them to iGEM parts registry with detailed characterization. See more at <a href="https://2016.igem.org/Team:OUC-China/Part_Collection">parts</a>.</p>
 +
<br id="float04">
 +
<hr>
 +
<h3 class="text-center">CONSTRUCT TOOLKIT</h3>
 +
<p>V. Constructed a set of <b>toolkit</b> of stem-loops with different folding free energy.</p>
 
<div class="row">
 
<div class="row">
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
<div class="col-md-8">
 
<div class="col-md-8">
−
<img src="https://static.igem.org/mediawiki/2016/3/31/T--OUC-China--pr-3-7.jpg" class="img-responsive" alt="Structures of three discovered stem-loops">
+
<img src="https://static.igem.org/mediawiki/2016/3/30/T--OUC-China--proof-444.png" class="img-responsive" alt="toolkit">
 
</div>
 
</div>
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
</div>
 
</div>
−
<p style="font-size:16px;">Figure 6 Relative expression on RNA and protein level with stem-loop of -14.9 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
+
<div class="clearfix"></div>
 
<div class="row">
 
<div class="row">
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
<div class="col-md-8">
 
<div class="col-md-8">
−
<img src="https://static.igem.org/mediawiki/2016/e/e8/T--OUC-China--pr-3-8.jpg" class="img-responsive" alt="Structures of three discovered stem-loops">
+
<img src="https://static.igem.org/mediawiki/2016/7/79/T--OUC-China--proof-555.png" class="img-responsive" alt="toolkit">
 
</div>
 
</div>
 
<div class="col-md-2"></div>
 
<div class="col-md-2"></div>
 
</div>
 
</div>
−
<p style="font-size:16px;">Figure 6 Relative expression on RNA and protein level with stem-loop of -30.1 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
+
<p style="font-size:16px;">Figure 4 Detailed data of designed stem-loops with various folding free energy in our toolkit. <a href="https://2016.igem.org/Team:OUC-China/Results#float04">Click here</a> to know more.</p>
−
<div class="row">
+
<br id="float05">
−
<div class="col-md-2"></div>
+
<hr>
−
<div class="col-md-8">
+
<h3 class="text-center">MODEL</h3>
−
<img src="https://static.igem.org/mediawiki/2016/0/0c/T--OUC-China--pr-3-9.jpg" class="img-responsive" alt="Structures of three discovered stem-loops">
+
<p>VI. Model the process of mRNA degradation in the present of stem-loops. See more at <a href="https://2016.igem.org/Team:OUC-China/Model">model</a>.</p>
−
</div>
+
<br id="float06">
−
<div class="col-md-2"></div>
+
<hr>
−
</div>
+
<h3 class="text-center">SOFTWARE</h3>
−
<p style="font-size:16px;">Figure 6 Relative expression on RNA and protein level with stem-loop of -34.4 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.</p>
+
<p>VII. Create software to realize sequence scanning and stem-loop predicting based on our model. See more at <a href="https://2016.igem.org/Team:OUC-China/Software">software</a>.</p>
−
<p>These parts are also have fine regulatory effects Combine those validated previous stem-loops and our designed ones, we got a set of stem-loops.</p>
+
−
 
+
−
<br />
+
 
</div>
 
</div>
 
</div>
 
</div>
Line 274: Line 259:
 
<p>1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences</p>
 
<p>1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences</p>
 
<p>2.NEW ENGLAND Biolabs</p>
 
<p>2.NEW ENGLAND Biolabs</p>
−
<p>3.Genscript</p>
+
<p>3.GenScript</p>
 
</div>
 
</div>
 
<div class="col-md-5 Contact">
 
<div class="col-md-5 Contact">

Revision as of 02:27, 20 October 2016

Proof

project-banner


NATIVE STEM-LOOPS

I. Validate that native stem-loops can affect the expression of its flanking cistron based on our platform Click here to know more.

native stem-loops

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.

II. Prove that native stem-loops with gradient folding free energy results in differential expression. Click here to know more.

native stem-loops

Figure 2 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.


DESIGNED STEM-LOOPS

III. Verify the regulatory effect of designed stem-loops.

designed stem-loops

Figure 3 One of the designed stem-loop of -34.4 kcal/mol (measured by Mfold) inserted into intergenic regions can also tune the expression of gfp and mcherry as expected, similarly with the effect of native stem-loops. Error bars indicate s.d. of mean of experiments in triplicate.


STANDARDIZE PARTS

IV. Standardize measured regulatory elements and submit them to iGEM parts registry with detailed characterization. See more at parts.


CONSTRUCT TOOLKIT

V. Constructed a set of toolkit of stem-loops with different folding free energy.

toolkit
toolkit

Figure 4 Detailed data of designed stem-loops with various folding free energy in our toolkit. Click here to know more.


MODEL

VI. Model the process of mRNA degradation in the present of stem-loops. See more at model.


SOFTWARE

VII. Create software to realize sequence scanning and stem-loop predicting based on our model. See more at software.

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

Designed and built by @ Jasmine Chen and @ Zexin Jiao

We are OUC-iGEM logo-one logo-two