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

 
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<li class="active"><a href="#float01">Background</a></li>
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<li class="active"><a href="#float01"><span style="font-family:'Lucida Calligraphy';font-size:22px;">W</span>here we started</a></li>
<li><a href="#float02">Design</a></li>
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<li><a href="#float02"><span style="font-family:'Lucida Calligraphy';font-size:22px;">L</span>ook closely to the existing strategies</a></li>
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<li><a href="#float03"><span style="font-family:'Lucida Calligraphy';font-size:22px;">I</span>nspiration from Nature</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">F</span>ocus on the deep mechanism</a></li>
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<li><a href="#float04"><span style="font-family:'Lucida Calligraphy';font-size:22px;">O</span>ur creative design</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">Background<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png"></h3>
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<br>
<p>Nowadays,quantitative has become a general trend, and of which the control of gene expression play a vital role. Until now, there’re several ways to control gene expression and consequently achieve stoichiometry and functional protein products.</p>
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<h3 class="text-center">WHERE WE STARTED</h3>
<p>Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene  products(protein or RNA). Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein.</p>
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<p>In synthetic biology, microorganisms with modified metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway[1,2]. Indeed, pathway optimization requires to adjust expression of multiple genes at appropriately balanced levels, for example the synthesic of poly(3-hydroxybutyrate)[3] and Mevalonate[4].Similarly, manipulation of multisubunit proteins (for example F1F0-ATPase ) also requires coordinated expression of several genes to produce the subunits at the appropriate stoichiometries[5]. As is done in the prokaryotes, grouping a cluster of gene s into a single polycistron is a convenient mean 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>
                                <b style="font-size:18px">Modification of DNA(左侧插图~~~~)</b>
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                                <p>In eukaryotes, the modification of DNA’s chromatin structure, such as histone modifications directed by DNA methylation[1], ncRNA, or DNA-binding protein, may up or down regulate the expression of a gene.</p>
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                                <b style="font-size:18px">Regulation of transcription(左侧插图~~~~)</b>
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                                <p>This controls when transcription occurs and the amount of RNA be created. The mechanisms usually includes specificity factors, general transcription factors, repressors, activators, enhancers [2] and silencers.</p>
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                                <b style="font-size:18px">Post-transcriptional regulation [3] (左侧插图~~~~)</b>
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                                <p>In eukaryotes, there have some mechanisms on how much the mRNA is translated into proteins [4]. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript.</p>
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                                <b style="font-size:18px">Regulation of translation(左侧插图~~~~)</b>
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                                <p>The translation of mRNA can also be controlled by a number of mechanisms[5], mostly at the level of initiation. The secondary structure of mRNA, antisense RNA binding, or protein binding[6] can all modulate the recruitment of the small ribosomal subunit. </p>
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                                <p>So many regulation methods have developed to achieve gene expression on desired level. Accurate as they are, they can hardly control the relative expression of several cistrons simultaneously. They are usually performed on operon level and may not have difference influence on each cistron.</p>
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                                <p>But on certain condition, it’s of vital importance to realize differential gene expression in polycistrons. For example, <a href="https://2014.igem.org/Team:Imperial">Team Imperial 2014</a> aimed to biosynthesize bacterial cellulose in E.coil, they need to transform two cistrons of different expression level into E.coil. Without efficient approaches, they did lots of jobs. They selected proper copied plasmid among 9 plasmids and then measured 15 Anderson promotors of different strength and finally selected a proper combination for the two different cistrons. </p>
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                                <p>This year, OUC-iGEM team devoted to exploring a novel regulation method on post-transcriptional level to realize differential expression in a polycistron. Details see the design part.</p>
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<h3 class="text-center">LOOK CLOSELT TO THE EXISTING STRATEGIES</h3>
<img src="https://static.igem.org/mediawiki/2016/8/84/T--OUC-China--project-2.3.png" class="img-responsive">
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<p>As a matter of fact, regulation of gene expression includes a wide range of strategies that are used by synthetic scientists, for examples copy number, promoters and RBS[6]. Despite their prominent advantages, it is nearly impossible to predict the necessary strengths of the promoters and ribosome binding sites (RBSs) to balance and coordinate the expression of multiple genes[7].What’s more, 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[8].</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">Design<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png"></h3>
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<p>For the public, we tried to publicize synthetic biology using relatively easy language toward as many people as possible through our activities. We went to communities and tourist attractions and met people from the young to the old. With the help of our brochures, we explained basic knowledge of synthetic biology and explained the meaning of quantification in their daily lives. They then realized that basic necessities of life were closely linked to quantification, like dietary recipe. We also posted our video on website which used simple language and vivid flash to introduce what is synthetic biology and how it can make our life better. </p>
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<img src="https://static.igem.org/mediawiki/2016/8/8b/T--OUC-China--pr-2-1.png" class="img-responsive" alt="Regulatory strategies">
<img src="https://static.igem.org/mediawiki/2016/9/9a/T--OUC-China--hp1.jpg" class="img-responsive">
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<p>Ergo, we are enlightened to try a rational design of one regulatory element to mainly reduce the number of trials and transform it into a user-friendly kit. We mainly focus on the prior design of the DNA sequences to work on the post- transcriptional level which can directly determine the relative levels of gene expression. Thus, stem-loop catches our attention.</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">Reference<img src="https://static.igem.org/mediawiki/2016/f/f8/T--OUC-China--head-icon2.fw.png"></h3>
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<h3 class="text-center">INSPIRATION FROM NATURE</h3>
<p>Specific to people working on science and technology industry, we tried to promoting synthetic biology in a deeper way. Therefore, we held academic lectures in Qingdao Association for Science and Technology for a delegation from Tibet, China. Over 90 teachers participated in lectures and they had more knowledge about synthetic biology. Moreover, due to coming from Tibet where the economic and educational development is relatively backward, these teachers could bring the conception of synthetic biology to Tibet and promoted it in these remote regions.</p>
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<p>Nature is uncanny workmanship. Recently, in <i>C. cellulolyticum</i>, within an polycistron encoding  cellulosome, Xu reported that 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[9]. Thus we consider the possibility of stem loops inserted in the intergenic region for tuning expression in <i>E.coli</i> which is more 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>[10], confirmed our thinking. Thus, we decided to develop stem loops with different free folding energy inserted in the intergenic region of two genes to coordinate expressions.</p>
<img src="https://static.igem.org/mediawiki/2016/f/f4/T--OUC-China--hp2.jpg" class="img-responsive">
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<h3 class="text-center">FOCUS ON THE DEEP MECHANISM</h3>
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<h3 class="text-center">Cistrons Concerto</h3>
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<p>The operon is transcribed by its sole promoter and the primary transcript is cleaved into several secondary transcripts by RNase E, a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence . However, the stability of these secondary transcripts against exonuclease degradation from the 3’ end varied due to their distinct terminal structure. When stem loops inserted in the 3’ end of the upstream gene, it protects its mRNA against the cleavage of exonuclease, increasing the ratio of abundance of the first gene product relative to that of the second gene product. Furthermore, the lower free energy of stem loops are, the more stable the secondary transcripts of the upstream are, tuning the expression of multiple genes.</p>
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<h3 class="text-center">OUR CREATIVE DESIGN</h3>
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<p>For further developing stem loops as useful regulatory parts, we racked our brains and  divided our project into two parts. First we exploited stem loops as new parts to tune the expression and transformed it into a user-friendly toolkit. SOLID data are carried out to confirmed its function. And then we explored more of this novel regulation method though modeling and software. More details in <a href="https://2016.igem.org/Team:OUC-China/Design">Design</a></p>
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<h3 class="text-center">FURTHER APPLICATION</h3>
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<p>After <a href="https://2016.igem.org/Team:OUC-China/Potential_application">visiting some local industrial enterprises</a>, we aimed to devoted our design into a practical industrial production, for improving product titers, yields, and productivity. In the fermentation of beer, the regulation of GSH, a reducing substance that is resistant to beer aging, contributes to increase the productivity of beer. GSH is composed of two subunits GSHⅠand GSHⅡ. Regulating the ratio of GSHⅠand GSHⅡ at proper stoichiometries results in higher yields . Thus, we are going to insert the stem loops with required free folding energy into the coding genes GSHⅠand GSHⅡto coordinate their relative expressions.</p>
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<h4 class="text-center">REFERENCE</h4>
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<p style="font-size:16px;">[1]. Khosla, C. & Keasling, J.D. Metabolic engineering for drug discovery and development.Nat. Rev. Drug Discov. 2, 1019–1025 (2003).<br>[2]. 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>[3]. 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>[4]. 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>[5]. White, M.M. Pretty subunits all in a row: using concatenated subunit constructs to force the expression of receptors with defined stoichiometry and spatial arrangement. Mol. Pharmacol. 69, 407–410 (2006).<br>[6]. 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>[7]. Pfleger B F, Pitera D J, Smolke C D, et al. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes[J]. Nature biotechnology, 2006, 24(8): 1027-103<br>[8]. 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>[9]. 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>[9]. 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.<br>[10]. Mackie, George A. "RNase E: at the interface of bacterial RNA processing and decay." Nature Reviews Microbiology 11.1 (2013): 45-57.<br>[10]. Liao X Y, Shen W, Chen J, et al. Improved glutathione production by gene expression in Escherichia coli[J]. Letters in Applied Microbiology, 2006, 43(2):211-214.</p>
 
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<h3>Thanks:</h3>
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<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>3.Genscript</p>
 
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Latest revision as of 20:44, 19 October 2016

Introduction

project-banner


WHERE WE STARTED

In synthetic biology, microorganisms with modified metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway[1,2]. Indeed, pathway optimization requires to adjust expression of multiple genes at appropriately balanced levels, for example the synthesic of poly(3-hydroxybutyrate)[3] and Mevalonate[4].Similarly, manipulation of multisubunit proteins (for example F1F0-ATPase ) also requires coordinated expression of several genes to produce the subunits at the appropriate stoichiometries[5]. As is done in the prokaryotes, grouping a cluster of gene s into a single polycistron is a convenient mean 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



LOOK CLOSELT TO THE EXISTING STRATEGIES

As a matter of fact, regulation of gene expression includes a wide range of strategies that are used by synthetic scientists, for examples copy number, promoters and RBS[6]. Despite their prominent advantages, it is nearly impossible to predict the necessary strengths of the promoters and ribosome binding sites (RBSs) to balance and coordinate the expression of multiple genes[7].What’s more, 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[8].

Regulatory strategies

Ergo, we are enlightened to try a rational design of one regulatory element to mainly reduce the number of trials and transform it into a user-friendly kit. We mainly focus on the prior design of the DNA sequences to work on the post- transcriptional level which can directly determine the relative levels of gene expression. Thus, stem-loop catches our attention.



INSPIRATION FROM NATURE

Nature is uncanny workmanship. Recently, in C. cellulolyticum, within an polycistron encoding cellulosome, Xu reported that 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[9]. Thus we consider the possibility of stem loops inserted in the intergenic region for tuning expression in E.coli which is more widely utilized as an engineered strain.
Fortunately, Keasling has identified that stem loops function well in the post-transcriptional process in E.coli[10], confirmed our thinking. Thus, we decided to develop stem loops with different free folding energy inserted in the intergenic region of two genes to coordinate expressions.



FOCUS ON THE DEEP MECHANISM

Mechanism model

The operon is transcribed by its sole promoter and the primary transcript is cleaved into several secondary transcripts by RNase E, a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence . However, the stability of these secondary transcripts against exonuclease degradation from the 3’ end varied due to their distinct terminal structure. When stem loops inserted in the 3’ end of the upstream gene, it protects its mRNA against the cleavage of exonuclease, increasing the ratio of abundance of the first gene product relative to that of the second gene product. Furthermore, the lower free energy of stem loops are, the more stable the secondary transcripts of the upstream are, tuning the expression of multiple genes.



OUR CREATIVE DESIGN

For further developing stem loops as useful regulatory parts, we racked our brains and divided our project into two parts. First we exploited stem loops as new parts to tune the expression and transformed it into a user-friendly toolkit. SOLID data are carried out to confirmed its function. And then we explored more of this novel regulation method though modeling and software. More details in Design

Project idea


FURTHER APPLICATION

After visiting some local industrial enterprises, we aimed to devoted our design into a practical industrial production, for improving product titers, yields, and productivity. In the fermentation of beer, the regulation of GSH, a reducing substance that is resistant to beer aging, contributes to increase the productivity of beer. GSH is composed of two subunits GSHⅠand GSHⅡ. Regulating the ratio of GSHⅠand GSHⅡ at proper stoichiometries results in higher yields . Thus, we are going to insert the stem loops with required free folding energy into the coding genes GSHⅠand GSHⅡto coordinate their relative expressions.



REFERENCE

[1]. Khosla, C. & Keasling, J.D. Metabolic engineering for drug discovery and development.Nat. Rev. Drug Discov. 2, 1019–1025 (2003).
[2]. 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).
[3]. 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.
[4]. 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.
[5]. White, M.M. Pretty subunits all in a row: using concatenated subunit constructs to force the expression of receptors with defined stoichiometry and spatial arrangement. Mol. Pharmacol. 69, 407–410 (2006).
[6]. 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.
[7]. Pfleger B F, Pitera D J, Smolke C D, et al. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes[J]. Nature biotechnology, 2006, 24(8): 1027-103
[8]. 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
[9]. 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.
[9]. 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.
[10]. Mackie, George A. "RNase E: at the interface of bacterial RNA processing and decay." Nature Reviews Microbiology 11.1 (2013): 45-57.
[10]. Liao X Y, Shen W, Chen J, et al. Improved glutathione production by gene expression in Escherichia coli[J]. Letters in Applied Microbiology, 2006, 43(2):211-214.

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