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

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<li><a href="https://2016.igem.org/Team:OUC-China/Project">Project</a></li>
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<li><a href="https://2016.igem.org/Team:OUC-China/Collaborations">Collaborations</a></li>
 
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<h3 class="text-center">ABSTRACT</h3>
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<p class="text-center">As is known, once forming stem-loops, the oligonucleotides will be more stable than the single-stranded ones. And mRNA with stem-loop at its 3’ or 5’ end often get a longer lifetime than the linear one owe to the stem-loop’s resistance to exonuclease. Our team tend to design a series of stem-loops each followed by the same endonuclease site and are transcribed as one polycistron. Once digested by endonuclease and seperate into several independent fragments, cistrons with different ∆G stem-loops will get different stability, thus influence the amount of expressed proteins. In this way, we can decouple the expression level of upstream and downstream genes of the same operon by simply designing different stem-loops. Futhermore, with quantitative ∆G of stem-loops, we even can achieve the ratio expression of target proteins. It is a creative regulating method and we attempt to provide a series of standard regulation parts for others.</p>
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<p>In synthetic biology, microorganisms with modified metabolic pathways are employed as a reaction vessel to synthesis natural or unnatural products. So it involves the introduction of several genes encoding the enzymes of a metabolic pathway  . Indeed, pathway optimization requires to adjust expression of multiple genes at appropriately balanced levels for higher production , for example the synthesis of poly(3-hydroxybutyrate)  and Mevalonate  . As is done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient means for regulating genes simultaneously. Thus, for the sake of tuning the expressions of genes within polycistrons, we aim to develop a tightly regulated, predictable components –stem loop to realize cistron concerto.</p>
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<p> Several prominent strategies have been employed to regulate differential expression of serial genes such as copy number, promoters and RBS .  Compared to their obvious advantages, they are limited by high-flux design. When traditional cloning is utilized, constructing libraries of hundreds of configurations of pathway genes with varying copy number, promoter, and RBS strengths is a daunting and time consuming task even for small pathways . Ergo, we are inspired to have a nice try about rational design of one regulated element to reduce the number of trials and transform it into a user-friendly kit. With brainstorms and information collection, we turn to focus on the prior design of the DNA sequences to work on the post- transcriptional level for it can directly determine the relative levels of gene expression.</p>
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<h3>3. Inspiration from Nature</h3>
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<p>In <i>C. cellulolyticum</i>, within an polycistron encoding cellulosome, stem-loop structures associated with those intergenic post-transcriptional processed sites located at 3’ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes.  . Thus we consider the possibility of stem loops inserted in the intergenic region for tuning expression in <i>E.coli</i> which has been widely utilized as an engineered strain.<br>Fortunately, Keasling has identified that stem loops function well in the post-transcriptional process in <i>E.coli</i> . And he confirmed that TIGR(tunable intergenic regions) sequences generally had a stronger influence on the expression of the gene 3’to the TIGR than on the gene 5’ to the TIGR. <br>Thus, we decided to develop stem loops within intergenic region as a regulatory elements to coordinate expressions.</p>
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<h3>4. Our story</h3>
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<p> Khosla, C. & Keasling, J.D. Metabolic engineering for drug discovery and development. Nat. Rev. Drug Discov. 2, 1019–1025 (2003).<br>Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).<br>Li T, Ye J, Shen R, et al. Semi-rational approach for ultra-high poly (3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening[J]. ACS synthetic biology, 2016.<br>Dudley Q M, Anderson K C, Jewett M C. Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis[J]. ACS Synthetic Biology, 2016.<br>Song C W, Lee J, Ko Y S, et al. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid[J]. Metabolic engineering, 2015, 30: 121-129.<br>Jones, J.A., O.D. Toparlak, and M.A. Koffas, Metabolic pathway balancing and its role in the production of biofuels and chemicals. Curr Opin Biotechnol, 2015. 33: p. 52-9<br>Xu C, Huang R, Teng L, et al. Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization[J]. Nature communications, 2015, 6.<br>Smolke C D, Keasling J D. Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon[J]. Biotechnology and bioengineering, 2002, 80(7): 762-776.</p>
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<a href="#"><img src="https://static.igem.org/mediawiki/2016/4/41/T--OUC-China--menu-icon-1.fw.png" class="img-responsive" alt="Purpose"><p class="text-center">Purpose</p></a>
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<a href="#"><img src="https://static.igem.org/mediawiki/2016/1/1d/T--OUC-China--menu-icon-2.fw.png" class="img-responsive" alt="Principle"><p class="text-center">Principle</p></a>
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<a href="https://2016.igem.org/Team:OUC-China/Attributions"><img src="https://static.igem.org/mediawiki/2016/8/8a/T--OUC-China--menu-icon-4.fw.png" class="img-responsive" alt="Attrbution"><p class="text-center">Attrbutions</p></a>
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<p style="margin-top:20px;">Thanks to:<img src="https://static.igem.org/mediawiki/2016/5/57/T--OUC-China--foot1.jpg" style="margin-left:10px;" alt="Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences"><img src="https://static.igem.org/mediawiki/2016/f/f0/T--OUC-China--foot2.jpg" style="margin-left:10px;" alt="Biolabs"></p>
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Revision as of 10:26, 17 October 2016

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ABSTRACT

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As is known, once forming stem-loops, the oligonucleotides will be more stable than the single-stranded ones. And mRNA with stem-loop at its 3’ or 5’ end often get a longer lifetime than the linear one owe to the stem-loop’s resistance to exonuclease. Our team tend to design a series of stem-loops each followed by the same endonuclease site and are transcribed as one polycistron. Once digested by endonuclease and seperate into several independent fragments, cistrons with different ∆G stem-loops will get different stability, thus influence the amount of expressed proteins. In this way, we can decouple the expression level of upstream and downstream genes of the same operon by simply designing different stem-loops. Futhermore, with quantitative ∆G of stem-loops, we even can achieve the ratio expression of target proteins. It is a creative regulating method and we attempt to provide a series of standard regulation parts for others.

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Purpose

Purpose
Principle

Principle
Potential

Potential
Attrbution

Attrbutions
Team

Team
Human Practices

Human Practices

Cistrons Concerto

Thanks


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

2.NEW ENGLAND Biolabs

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