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

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<li class="dropdown"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project<span class="caret"></span></a>
 
<li class="dropdown"><a class="dropdown-toggle" data-toggle="dropdown" href="#">Project<span class="caret"></span></a>
 
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<li><a href="https://2016.igem.org/Team:OUC-China/Project">Introduction</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Description">Description</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Design">Design</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Design">Design</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Proof">Proof of concept </a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Proof">Proof of concept </a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Human_Practices">Overview</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Human_Practices">Overview</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Communicating">Communicating & Improving</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Communicating">Communicating & improving</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Investigating">Investigating & Promoting</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Investigating">Investigating & promoting</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Potential_application">Potential application</a></li>
 
<li><a href="https://2016.igem.org/Team:OUC-China/Potential_application">Potential application</a></li>
 
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<li class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">O</span>verview</a></li>
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<li><a href="#float02">Preliminary experiments</a></li>
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<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">D</span>esigned stem-loops</a></li>
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<li><a href="#float03">Native stem loops</a></li>
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<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">S</span>tandardize parts</a></li>
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<li><a href="#float04">Native VS Designed stem-loops</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">C</span>onstruct toolkit</a></li>
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<h3 class="text-center"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">OVERVIEW<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
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<h3 class="text-center">TEST PREVIOUS STEM-LOOPS</h3>
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<br />
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<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>To explore this novel regulation method, we have several steps to go:<br>1. Employed preliminary experiment to test if the differential expression is caused by stem loop. <br>2. Predicted the protection effect using the native stem loops.<br>3. Tested the difference between the native and the designed stem loops.<br>4. Validated the primary relationship of free energy and quantitative expression using designed stem loops with gradient free energy.<br>5. Further we tested our result in the tri-fluorescent reporter system.</p>
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<p>The result see as follows:</p>
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<img src="" width="" height="" alt="" />
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<img src="https://static.igem.org/mediawiki/2016/5/5e/T--OUC-China--result1.png" class="img-responsive" alt="Folding -25.6">
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</div>
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<div class="col-md-2"></div>
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</div>
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<p style="font-size:16px;">Figure 1 Relative expression on transcriptional and translational level for stem-loops 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 gene. Fluorescence is normalized by OD600. . Error bars indicate s.d. of mean of experiments in triplicate</p>
 +
<p>After functions and sequence confirmed, we tested their function and standardized them to submitting 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.</p>
 +
<div class="row">
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<div class="col-md-2"></div>
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<img src="https://static.igem.org/mediawiki/2016/7/73/T--OUC-China--pr-3-4.jpg" class="img-responsive" alt="stem-loops-123">
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</div>
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<div class="col-md-2"></div>
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</div>
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<p style="font-size:16px;">Figure 2 Structures of three discovered stem-loops</p>
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<div class="col-md-2"></div>
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<img src="https://static.igem.org/mediawiki/2016/9/95/T--OUC-China--result3.jpg" class="img-responsive" alt="toolkit">
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<div class="col-md-2"></div>
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</div>
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<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>
 
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<h3 class="text-center" style="font-size:22px"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">PRELIMINARY EXPERIMENTS<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
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<h3 class="text-center">MEASURE A SERIES OF DESIGNED STEM-LOOPS</h3>
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<br />
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<p>In addition to the confirmation of previous discovered 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 same rules, we designed several stem-loops (Figure 4) with various free folding energy and tested them again (Figure 5). As shows, these parts also tune the ratio of the upstream and downstream genes.</p>
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<h4>Stem loops and Terminators</h4>
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<p>To achieve differentiated expression between up and down stream genes within a polycistron, we need to find out the mechanism that causes the difference. Is it caused by stem-loops? How does it realize? Is it caused by protecting the upstream gene，or by decreasing the downstream one just like terminators?</p>
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<p><i>Escherichia coli</i> rho-independent transcription terminators are characterized by an RNA structure with a GC-rich stem-loop followed by a series of uridine residues, which is exactly similar to our designed stem loops. However, it is supposed that our designed stem loops work through interfering in the degrading process by resisting to the exoribonuclease instead of terminating the downstream gene. Thus, we constructed circuits to test if the expression of the downstream gene was reduced owing to the stem loops. We inserted a stem loop between two reporters contrast to the control one without stem loops in the intergenic region. Then we measured the reporting proteins both on the mRNA and the protein level. The result are as follows:</p>
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<img src="https://static.igem.org/mediawiki/2016/d/d5/T--OUC-China--pr-3-6.jpg" class="img-responsive" alt="1">
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<p>Figure 3: The figure shows the fluorescent of downstream mCherry in the circuit with or without stem-loop, and there’s no significant difference between them(P=0.01). Error bars indicate s.d. of mean of all sequences.</p>
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<p style="font-size:16px;">Figure 4 Structure of three stem-loops designed by ourselves</p>
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<h4>Gene sequence</h4>
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<p>We aimed to develop this regulation method into a toolkit that can be applied to other polycistrons, not only the dual-fluorescent system. So we had to make sure that the dual-fluorescent reporter system we constructed didn’t influence our result. In other words, it was the stem-loop itself that generate this kind difference. So we swapped the location of GFP and mCherry and constructed the following circuit to test it.</p>
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<p style="font-size:16px;">Figure 5 Relative expression on RNA and protein level for designed stem-loop of -30.1, -34.4,38.8,44.9kcal/mol (measured by Mfold) contrast to the control group with no 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>
 
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<h3 class="text-center"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">NATIVE STEM LOOPS<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
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<h3 class="text-center">CONSTRUCT A WONDERFUL TOOLKIT</h3>
−
<br />
+
<p>Collected those validated previous stem-loops and our designed ones, we got a set of stem-loops forming a toolkit. Below are the basic information of stem-loops in our toolkit.</p>
−
<p>It is reported that there are several native stem loops that may have effects on its flanking genes, either at the 3’ termini or the 5’ termini[1]. Ergo, we use two native stem loops from <i>R. capsulatus</i> and <i>E.coli[2]</i> 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.</p>
+
<div class="row">
−
<img src="" width="" height="" alt="" />
+
<div class="col-md-2"></div>
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<p>Figure 7: The protein fluorescence of upstream GFP to downstream mCherry of different circuits, background subtraction has been normalized with control group.Ratio  = {[RFPterm/GFPterm]/[(RFPcontrol/GFPcontrol)mean][3]</p>
+
<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">
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</div>
 +
<div class="col-md-2"></div>
 +
</div>
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<p style="font-size:16px;">Figure 4 Structure of three stem-loops designed by ourselves</p>
 
<br id="float04">
 
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<hr>
 
<hr>
−
<h3 class="text-center" style="font-size:18px"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">NATIVE VS DESIGNED STEM-LOOPS<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
+
<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 are the relative expression on two levels of different stem-loops in our toolkit.</p>
−
<p> By using the native stem loop, we have confirmed that in <i>E.coli</i>, the stem loop at the 3’termini can indeed influence the quantitative expression of its upstream gene. Next we aimed to design nonnative stem loops to verify the precise correlation between the △G and the quantitative expression. But only if this mechanism is determined by △G can we design the stem-loops quantitatively. Thus we need to explore whether the protecting efficiency of the stem loops is determined by its Gibbs free energy or by other factors such as certain specific sequence.</p>
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<div class="row">
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<p>Then we designed 3 stem loops that have the same free energy as a native one (△G=-38.7kcal/mol)[4] but with different base sequence and measured their relative expression of the up and down stream genes on protein and mRNA level.</p>
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<h3 class="text-center" style="font-size:22px"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">THE PRECISE CORRELATION<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
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<p style="font-size:16px;">Figure 6  RNA varying with folding free energy of stem-loops</p>
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<br />
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<div class="clearfix"></div>
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<p>We designed a series of <a href="">stem loops</a> of gradient free energy to explore the relationship between free energy and quantitative expression. And measured the relative expression of the up and down stream genes on both mRNA and protein level. The result are as follows:</p>
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<h3 class="text-center"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--OUC-China--head-icon1.fw.png" alt="icon">FURTHER VERIFICATION<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png" alt="icon"></h3>
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<br />
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</div>
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<p>After we got the relationship between free energy and quantitative expression, we wanted to test our result in the tri-fluorescent reporter system.and we constructed the tri-fluorescent reporter system as follows:</p>
+
<p style="font-size:16px;">Figure 7  protein expression varying with folding free energy of stem-loop</p>
−
<img src="" width="" height="" alt="" />
+
<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>
−
<p>The result are as follows:</p>
+
−
<p style="font-size:16px;">[1] Carrier, T. A., & Keasling, J. D. (1997). Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5′ cassette system.Biotechnology and bioengineering, 55(3), 577-580.<br>[2] Smolke, C. D., & Keasling, J. D. (2002). Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng, 80(7), 762-776. doi: 10.1002/bit.10434<br>[3] Nojima, Takahiko, et al. "Controlling the expression ratio of two proteins by inserting a terminator between the two genes." Nucleic Acids Symposium Series. Vol. 50. No. 1. Oxford University Press, 2006.<br>[4] Nilsson, P., & Uhtin, B. E. (1991). Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing. Molecular microbiology, 5(7), 1791-1799.</p>
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<h3>Thanks</h3>
 
<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>
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<p>2.NEW ENGLAND Biolabs</p>
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<p>3.GenScript</p>
 
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Revision as of 03:16, 20 October 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 Relative expression on transcriptional and translational level for stem-loops 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 gene. Fluorescence is normalized by OD600. . Error bars indicate s.d. of mean of experiments in triplicate

After functions and sequence confirmed, we tested their function and standardized them to submitting 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.

stem-loops-123

Figure 2 Structures of three discovered 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 discovered 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 same rules, we designed several stem-loops (Figure 4) with various free folding energy and tested them again (Figure 5). As shows, these parts also tune the ratio of the upstream and downstream genes.

1

Figure 4 Structure of three stem-loops designed by ourselves

2

Figure 5 Relative expression on RNA and protein level for designed stem-loop of -30.1, -34.4,38.8,44.9kcal/mol (measured by Mfold) contrast to the control group with no 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

Collected those validated previous stem-loops and our designed ones, we got a set of stem-loops forming a toolkit. Below are the basic information of stem-loops in our toolkit.

1

Figure 4 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 are the relative expression on two levels of different stem-loops in our toolkit.

1

Figure 6 RNA varying with folding free energy of stem-loops

2

Figure 7 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

Designed and built by @ Jasmine Chen and @ Zexin Jiao

We are OUC-iGEM logo-one logo-two