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

 
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   <title>Project</title>
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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>
<li><a href="#float03">Reference</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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<br id="float02">
                                <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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<br>
                                <b style="font-size:18px">Regulation of transcription(左侧插图~~~~)</b>
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<h3 class="text-center">LOOK CLOSELT TO THE EXISTING STRATEGIES</h3>
                                <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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<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>
                                <b style="font-size:18px">Post-transcriptional regulation [3] (左侧插图~~~~)</b>
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<div class="row">
                                <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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<img src="https://static.igem.org/mediawiki/2016/8/8b/T--OUC-China--pr-2-1.png" class="img-responsive" alt="Regulatory strategies">
                                <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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</div>
                                <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 <i>E.coil</i>, they need to transform two cistrons of different expression level into <i>E.coil</i>. 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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<div class="col-md-2"></div>
                                <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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</div>
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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">INSPIRATION FROM NATURE</h3>
<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>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>
                                <b style="font-size:18px">Overview</b>
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<br id="float04">
<p>In C.Cellulolyticum,it is reported that a novel regulation method was employed to regulate the differential expression of multiple cistrons within the same operon[1]. And we came out the idea of exploiting this mechanism to a toolkit using for regulating differential and quantitative expression within a polycistron.</p>
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                                <p>The principle is as follows:</p>
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<img src="https://static.igem.org/mediawiki/2016/6/62/T--OUC-China--project-2.1.png" class="img-responsive">
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                                <p class="text-center" style="font-size:14px;">Figure 1. Diagram of the post-transcriptional regulation method</p>
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                                <p>In bacteria genomes, genes are frequently organized as an operon that mostly encodes protein complexes, which is an efficient means to regulate transcription of multiple genes simultaneously. It is firstly transcribed by its sole promoter into the primary mRNA.<br>And then, in <i>E.coli</i> cases, the processing is initiated via a primary cleavage by RNase E[2]. It is a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence and its cleavage signals locate in the intergenic regions. Primary transcript then will be split by RNase E into several secondary transcripts. [Fig.3]<br>Stability of these secondary transcripts varied widely due to their distinct terminal sequences that convey resistance to exonuclease degradation. In other words, stem loops with lower free energy can protect mRNA more from exoribonuclease degradation and stay longer in cells, thus the transcripts can  translate more proteins. <br>With this system, we can regulate multiple genes in one operon express on desired quantitative level, realize multiple cistrons express coordinately, just like a melodic cistrons concerto.<br>To achieve this, we constructed dual-fluorescent and tri-fluorescent reporter system and designed a series of stem-loops to insert into the reporter system to test this regulating method. Following are our basal designs. </p>
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                                <b style="font-size:18px">Dual-fluorescent reporter system</b>
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<p>We want to test the protection effect on the upstream gene from stem-loops, thus we primarily construct a dual-fluorescent reporter system. The circuit is as follows: </p>
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<img src="https://static.igem.org/mediawiki/2016/e/e1/T--OUC-China--project-2.2.png" class="img-responsive">
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                                <p class="text-center" style="font-size:14px;">Figure 2. Dual-fluorescent reporter</p>
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                                <p>The promoter is inducible with arabinose and the two fluorescent reporter gfp and mCherry with their RBS are two parts in the iGEM part library, numbered <a href="http://parts.igem.org/Part:BBa_I13500">BBa_I13500</a> and <a href="http://parts.igem.org/Part:BBa_J06602">BBa_J06602</a>, the RNase E site between them is from <i>E.coli</i> pap operon [3], a relative high efficient endonuclease cleavage site through experiments. The stem-loops are from the series of stem loops we designed.</p>
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                                <b style="font-size:18px">Stem-loops</b>
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<p>We designed a series of stem-loops of gradient free energy to test this regulation method. And we determined the free energy of stem-loops with RNA folding program (Mfold). [4][5]</p>
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                                <p>We designed stem-loops in the view of 3 parts: loop, stem and a tail ahead with a pair of CG as "sealing nucleotides", see as figure 3.</p>
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<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 class="text-center" style="font-size:14px;">Figure 3: Diagram of the designed stem-loops</p>
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                                <p>Loop: Since the loop makes little contribution to the whole free energy, we used the same loop throughout our designs. That is U-U-A-C-A-U-G-A-U-U. <br>Stem: The stem-loop's free energy is mainly decided by the pairing stem section. We chose the ratio of "A+U" to "C+G" as 1:1. For too many C/G may cause the energy too low and hard to adjust, whereas too many A/U are easy to cause terminating effect owing to the structure similarity to rho-independent terminators[6].. Choosing the ratio as 1:1, we can easily adjust the free energy by change the length of the paring stem. <br>"Sealing nucleotides": A pair of CG at the end of a stem-loop can help stabilizing the secondary structure by avoiding flanking extra base paring in different context circuit. <br>Following these principles, the stem-loops we designed are diaplayed in table 1, they are of gradient free energy:
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<img src="https://static.igem.org/mediawiki/2016/b/bc/T--OUC-China--project-2.4.jpg" class="img-responsive">
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                                <p>We measured these stem loops in the dual-fluorescent reporter system. By analyzing the amount of mRNA and protein expressed in different circuits, we aimed to explore the correlation between the folding free energy and the quantitative expression. We measured the quantitative expression on two levels: the transcriptional level and the translational level. On the transcriptional level, we aimed to find if the difference is actually caused by the varied mRNA degradation. Upon the translational level, we explored the possibility and prospect of this post-transcriptional regulation mechanism on practical application.</p>
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                                <b style="font-size:18px">Tri-fluorescent reporter system</b>
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                                <p>By the dual-fluorescent reporter system we obtain the correlation between  folding free energy and quantitative expression of mRNA and protein fluorescence. Then we constructed a tri-fluorescent reporter system to furtherly test this relationship. The circuit is as follows:</p>
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<img src="https://static.igem.org/mediawiki/2016/a/ac/T--OUC-China--project-2.5.png" class="img-responsive">
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                                <p class="text-center" style="font-size:14px;">Figure 4</p>
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                                <p>We use tri-fluorescent reporters to test the correlation we obtained, the inserted CFP with RBS is also a part in the part library, numbered <a href="http://parts.igem.org/Part:BBa_I13600">BBa_I13600.</a> <br>We use this circuit to measure the effect of two different stem loops, with  folding free energy of -51.4kcal/mol and -25.6kcal/mol. Detailed data see our <a href="https://2016.igem.org/Team:OUC-China/Results">result page</a>.</p>
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                                <b style="font-size:18px">Future</b>
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                                <p>This year, our team devoted to exploring a novel regulation method on the post-transcription level. Once the correlation is established and confirmed, we can apply this to tune the expression of polycistron of interest. To achieve our goals, we have 3 steps to go: </p>
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                                <p>a)Through our data analysis and modelling get the correlation. This step is finished in our lab with the basal circuit, the constructed fluorescent reporter system. <br>b)To apply this regulation method to practice using, we will develop it as a toolkit. Now we are exploiting a software to realize structure prediction base on the different folding free energy according to the target ratio.<br>c)Then we can employ our software a specific protein for expression optimizing. This can be used for industrial fermentation, so we are consulting specialists and connecting with related industries for advice.</p>
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<h3 class="text-center">FOCUS ON THE DEEP MECHANISM</h3>
<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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                                <b style="font-size:18px">Ref. in Background:</b>
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                                <p style="font-size:14px">[1].Bell JT, Pai AA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, Gilad Y, Pritchard JK (2011). "DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines". Genome Biology. 12 (1): <br>[2]. Austin S, Dixon R (Jun 1992). "The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent". The EMBO Journal. 11(6): 2219–28. <br>[3].Weaver, Robert J. (2007). "Part V: Post-transcriptional events". Molecular Biology. Boston: McGraw Hill Higher Education..<br>[4].Hu W, Coller J (2012). "What comes first: translational repression or mRNA degradation? The deepening mystery of microRNA function". Cell Res. 22 (9): 1322–4. doi:10.1038/cr.2012.80<br>[5].Kozak M (1999). "Initiation of translation in prokaryotes and eukaryotes". Gene. 234 (2): 187–208. doi:10.1016/S0378-1119(99)00210-3. PMID 10395892<br>[6].Malys N, McCarthy JEG (2010). "Translation initiation: variations in the mechanism can be anticipated". Cellular and Molecular Life Sciences. 68 (6): 991-1003. doi: 10.1007/s00018-010-0588-z.</p>
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<div class="col-md-8">
                                <b style="font-size:18px">Ref. in Design:</b>
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<img src="https://static.igem.org/mediawiki/2016/9/9e/T--OUC-China--pr-2-2.jpg" class="img-responsive" alt="Mechanism model">
                                <p style="font-size:14px">[1] Xu, C., Huang, R., Teng, L., Jing, X., Hu, J., Cui, G., . . . Xu, J. (2015). Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization. Nat Commun, 6, 6900. doi: 10.1038/ncomms7900.<br>[2] Carpousis, A. J., Luisi, B. F., & McDowall, K. J. (2009). Endonucleolytic initiation of mRNA decay in Escherichia coli. Progress in molecular biology and translational science, 85, 91-135.<br>[3]Bricker, A. L., & Belasco, J. G. (1999). Importance of a 5′ Stem-Loop for Longevity ofpapA mRNA in Escherichia coli. Journal of bacteriology, 181(11), 3587-3590.<br>[4] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction[J]. Nucleic acids research, 2003, 31(13): 3406-3415.<br><a href="http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form">[5] http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form</a><br>[6] Yarnell W S, Roberts J W. Mechanism of intrinsic transcription termination and antitermination[J]. Science, 1999, 284(5414): 611-615.</p>
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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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<img src="https://static.igem.org/mediawiki/2016/7/73/T--OUC-China--pr-2-3.png" class="img-responsive" alt="Project idea">
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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 class="text-center" style="margin-top:20px;">Cistrons Concerto</h3>
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        <h3 class="text-center">Cistrons Concerto</h3>
 
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<b>About:</b>
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<h3>Thanks:</h3>
<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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<p>1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences</p>
<p style="margin-top:20px;">Designed and built by @ Jasmine Chen and @ Zexin Jiao</p>
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<p>2.NEW ENGLAND Biolabs</p>
<p style="margin-top:20px;">Code licensed under Apache License v4.0</p>
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<p>3.Genscript</p>
 
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<h3>Contact us:</h3>
<b>Contact us:</b>
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<p><b>E-mail</b>: oucigem@163.com</p>
<p style="margin-top:20px;">E-mail: oucigem@163.com</p>
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<p><b>Designed and built</b> by @ Jasmine Chen and @ Zexin Jiao</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

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