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

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<ul class="dropdown-menu">
 
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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_&_improving">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_&_promoting">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 class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">T</span>est previous stem-loops</a></li>
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<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">P</span>reliminary experiments</a></li>
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<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">D</span>esign our stem-loops</a></li>
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<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">N</span>ative stem loops</a></li>
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<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">S</span>tem-loops Designed by Ourselves</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">N</span>ative VS Designed stem-loops</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">O</span>ur Toolkit</a></li>
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<li><a href="#float05"><span style="font-family:'Lucida Calligraphy';font-size:22px;">T</span>he precise correlation</a></li>
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<li><a href="#float06"><span style="font-family:'Lucida Calligraphy';font-size:22px;">F</span>urther verification</a></li>
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<img src="https://static.igem.org/mediawiki/2016/d/d2/T--OUC-China--btt.png" alt="Back to top">
 
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<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>
 
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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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<p>To explore this novel regulation method, we have several steps to go:<br>1. Employed preliminary experiment to test if 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>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>
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<p class="text-center" style="font-size:16px;">Figure 1 Dual-fluorescent reporter system</p>
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<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>
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<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>
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<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>
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<img src="" class="img-responsive" alt="Structures of three discovered stem-loops">
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<p class="text-center" style="font-size:16px;">Figure 3 Structures of three discovered stem-loops</p>
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<img src="" class="img-responsive" alt="Structures of three discovered stem-loops">
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<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>
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<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>
 
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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" style="font-size:22px">DESIGN OUR STEM-LOOPS</h3>
 
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<h4>Stem loops and Terminators</h4>
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<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>
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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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<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 class="text-center" style="font-size:16px;">Figure 5 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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<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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<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>
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<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>
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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>
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<img src="" class="img-responsive" alt="Structures of three discovered stem-loops">
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<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>
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</div>
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<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>
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<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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<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 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>
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<br />
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<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>
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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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<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>
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<img src="" width="" height="" alt="" />
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<p>The result are as follows:</p>
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<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>
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<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 17:15, 19 October 2016

Proof

project-banner

This year, we devoted to the exploitation of new regulatory parts and the exploration of a kind of novel regulatory method.



TEST PREVIOUS STEM-LOOPS


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 registried parts.

Dual-fluorescent reporter system

Figure 1 Dual-fluorescent reporter system

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:

Dual-fluorescent reporter system

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.

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.

Structures of three discovered stem-loops

Figure 3 Structures of three discovered stem-loops

Structures of three discovered stem-loops

Figure 4 Relative expression of 3 stem-loops with different folding free energy. Error bars indicate s.d. of mean of experiments in triplicate.

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.


DESIGN OUR STEM-LOOPS


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:

Structures of three discovered stem-loops

Figure 5 Structure of three stem-loops designed by ourselves

Structures of three discovered stem-loops

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.

Structures of three discovered stem-loops

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.

Structures of three discovered stem-loops

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.

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.


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