Team:ZJU-China/project.htm

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<p align="justify">In cells, engineered multi-enzyme pathways are common and are often physically and spatially organized, thus leading to the high output efficiency. But engineered synthetic pathways utilizing non-homologous enzymes often suffer from low efficiency of production caused by relative lack of spatial organization. RNA scaffold is designed to co-localize enzymes through interactions between binding domains on the scaffold and target peptides fused to each enzyme in engineered biological pathways <i>in vivo</i>. The scaffold allows efficient channeling of substrates to products over several enzymatic steps by limiting the diffusion of intermediates thus providing a bright future for solving the problem.</p>
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<p align="justify">In cells, engineered multi-enzyme pathways are common and are often physically and spatially organized, thus leading to the high output efficiency. But engineered synthetic pathways utilizing non-homologous enzymes often suffer from low efficiency of production caused by relative lack of spatial organization. <b class="orange">RNA scaffold is designed to co-localize enzymes through interactions between binding domains on the scaffold and target peptides fused to each enzyme in engineered biological pathways <i>in vivo</i></b>. The scaffold allows <b class="orange">efficient channeling of substrates to products</b> over several enzymatic steps by <b class="orange">limiting the diffusion of intermediates</b> thus providing a bright future for solving the problem.</p>
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<p align="justify">ZJU-China aims to design and realize tunable RNA scaffolds to accelerate biological pathways and control them on and off. In order to achieve the object, we added an aptamer structure on RNA scaffold as a switch to regulate biological pathways by micromolecular ligands. Then we can control the all-or-none binding relationship between the enzymes and the scaffold by the absence and the presence of a special ligand. </p>
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<p align="justify">ZJU-China aims to design and realize tunable RNA scaffolds to accelerate biological pathways and control them on and off. In order to achieve the object, we added an aptamer structure on RNA scaffold as a switch to regulate biological pathways by micromolecular ligands. Then we can <b class="orange">control the all-or-none binding relationship</b> between the enzymes and the scaffold by the absence and the presence of a special ligand. </p>
<p align="justify">&nbsp;</p>
<p align="justify">&nbsp;</p>
<p align="justify">We demonstrated RNA scaffold do make the split GFPs get closer and fluoresce. As was expected, the riboscaffold with a theophylline aptamer can be regulated by theophylline in the range of 0-0.5mM IPTG. A scaffold library was also desired. By changing the sequence of MS2 aptamer binding site, we made the fluorescent decreased. The mutations with different arm length decrease the fluorescent intensity of split GPF by extending the distance between two split GFP parts FA and FB. It provides a series of half-on scaffolds. </p>
<p align="justify">We demonstrated RNA scaffold do make the split GFPs get closer and fluoresce. As was expected, the riboscaffold with a theophylline aptamer can be regulated by theophylline in the range of 0-0.5mM IPTG. A scaffold library was also desired. By changing the sequence of MS2 aptamer binding site, we made the fluorescent decreased. The mutations with different arm length decrease the fluorescent intensity of split GPF by extending the distance between two split GFP parts FA and FB. It provides a series of half-on scaffolds. </p>
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<p align="justify">In cells, engineered multi-enzyme pathways are common and are often physically and spatially organized, thus leading to the high output efficiency. But engineered synthetic pathways utilizing non-homologous enzymes often suffer from low efficienty of production caused by relative lack of spatial organization. Thus important issue lies in the method to increase the efficiency of the multi-enzyme pathways. </p>
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<p align="justify">In cells, engineered multi-enzyme pathways are common and are often physically and spatially organized, thus leading to the high output efficiency. But engineered synthetic pathways utilizing non-homologous enzymes often suffer from low efficienty of production caused by relative lack of spatial organization. Thus important issue lies in the method to <b class="orange">increase the efficiency of the multi-enzyme pathways</b>. </p>
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<p align="justify">Protein scaffolds can be designed to make enzymes closed through interactions between binding domains on the scaffold and target peptides fused to each enzyme. However, protein scaffold is usually large, has limit binding sites, and is hard to be engineered in architecture. DNA can be designed to self-assemble in vitro into many and varied nanostructures. However, DNA scaffold is hard to be controlled and might cause some potential problems <i>in vivo</i>. By contrast, RNA scaffold shows great advantages. For instance, RNA is more flexible, whose structures are varied, thus leading to their ease to splice. RNA scaffold is able to be controlled and has a satisfactory regulating efficiency. RNA scaffold works fast, because it doesn’t need translation like protein scaffold. Camille J. Delebecque and his colleagues have designed and assembled RNA structures and used them to speed up the reaction of hydrogen production. And that is what our project based on.</p>
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<p align="justify">Protein scaffolds can be designed to make enzymes closed through interactions between binding domains on the scaffold and target peptides fused to each enzyme. However, protein scaffold is usually large, has limit binding sites, and is hard to be engineered in architecture. DNA can be designed to self-assemble in vitro into many and varied nanostructures. However, DNA scaffold is hard to be controlled and might cause some potential problems <i>in vivo</i>. By contrast, RNA scaffold shows great advantages. For instance, <b class="orange">RNA is more flexible</b>, whose structures are varied, thus leading to their ease to splice. RNA scaffold is able <b class="orange">to be controlled</b> and has a satisfactory regulating efficiency</b>. RNA scaffold <b class="orange">works fast</b>, because it doesn’t need translation like protein scaffold. Camille J. Delebecque and his colleagues have designed and assembled RNA structures and used them to speed up the reaction of hydrogen production. And that is what our project based on.</p>
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<p align="justify">&nbsp;</p>
<img src="https://static.igem.org/mediawiki/2012/b/b7/Zju_Backround_syn_and_bio.png" width="700px" />
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<p class="fig" align="justify"><b>Fig.1</b>  How RNA scaffold works. FA and FB represent two halves of EGFP. FA and MS2 are connected with a linker of 30bp. FB and PP7 did the same. The purple scaffold is scaffold D0. MS2 and PP7 can specifically bind to two stem-loops on scaffold, thus FA and FB get closer and fluoresce under excitation of 480nm.</p>
<p class="fig" align="justify"><b>Fig.1</b>  How RNA scaffold works. FA and FB represent two halves of EGFP. FA and MS2 are connected with a linker of 30bp. FB and PP7 did the same. The purple scaffold is scaffold D0. MS2 and PP7 can specifically bind to two stem-loops on scaffold, thus FA and FB get closer and fluoresce under excitation of 480nm.</p>
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<h5>1). pCJDFA: FA-MS2 cloned into T7 duet expression vectors pACYCDuet-1  Spr</h5>
<h5>1). pCJDFA: FA-MS2 cloned into T7 duet expression vectors pACYCDuet-1  Spr</h5>
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<h5>2) pCJDFB (FB-PP7 cloned into T7 duet expression vector pCOLADuet-1)  Kanr</h5>
<h5>2) pCJDFB (FB-PP7 cloned into T7 duet expression vector pCOLADuet-1)  Kanr</h5>
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<h5>3) pCJDD0 (Scaffold D0 cloned into T7 duet expression vector PETDuet)  Ampr</h5>
<h5>3) pCJDD0 (Scaffold D0 cloned into T7 duet expression vector PETDuet)  Ampr</h5>
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<p><h5>4) BL21-star(DE3)</h5>  
<p><h5>4) BL21-star(DE3)</h5>  
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<p align="justify">Wash the bacteria twice with equivalent PBS. Then test the Fluorescence intensity (FI) and OD with Biotek Synergy Hybrid Reader.
<p align="justify">Wash the bacteria twice with equivalent PBS. Then test the Fluorescence intensity (FI) and OD with Biotek Synergy Hybrid Reader.
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<p align="justify">Data was shown in Fig.3.  The fluorescence of different expression systems are pictured by Olympus fluoview fv1000 confocal laser scanning microscope ( Fig.2)<p>
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<p align="justify">Data was shown in Fig.3.  The fluorescence of different expression systems are pictured by Olympus fluoview fv1000 confocal laser scanning microscope (Fig.2)<p>
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<h2>Results</h2>
<h2>Results</h2>
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<p align="justify">See relative results in RESULTS S0: Basic Scaffold.<p>
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<p>Contrasted to the fluorescence intensity (FI) of the E.coli which only express FA-MS2 and FB-PP7 fusion proteins, the fluorescence intensity of the E.coli with scaffold D0 was obviously increased. Thus, it was possible for us to carry out our development and reformation of RNA scaffold.</p>
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<p class="fig"><b>Fig.2</b> FI of Split GFPs without or with RNA scaffold. A.  BL21*(DE3) transformed with pCJDFA and pCJDFB.  B. BL21*(DE3) transformed with pCJDFA, pCJDFB and pCJDD0. The contrast of FI obviously shown that RNA scaffold D0 could bind split GFPs together, so that split GFPs could fluoresce. (Pictures were obtained with Olympus fluoview fv1000 confocal laser scanning microscope, using a 60X objective.)</p>
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<p class="fig"><b>Fig.3</b>  FI/OD of different transformation groups.  There exist significant differences among three groups. And as expected, split GFPs with scaffold D0 together can fluoresce stronger than those without scaffold. </p>
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<h3>Reference:</h3>
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<p class="ref">1. Thodey, K. & Smolke, C.D. Bringing It Together with RNA. Science 333, 412-413 (2011).</br>
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2. Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A. Organization of Intracellular Reactions with Rationally Designed RNA Assemblies. Science 333, 470-474 (2011).</p>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_1.htm">1. Summary</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_1.htm" style="text-decoration:none">1. Summary</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_2.htm">2. Design</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_2.htm" style="text-decoration:none">2. Design</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_4.htm" style="text-decoration:none">3. Preparation: Characterize previous parts</a>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_4.htm">4. Preparation: Characterize previous parts</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_3.htm" style="text-decoration:none">4. Characterization</a><br>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_5.htm">5. Results</a>
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<a target="brainFrame" href="https://2012.igem.org/Team:ZJU-China/project_s1_5.htm" style="text-decoration:none">5. Results</a>
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<p class="fig"><b>fig 1b.</b> The result of arm length mutating. Both D0M4 and D0M5 scaffold half-on GEP.</p>
<p class="fig"><b>fig 1b.</b> The result of arm length mutating. Both D0M4 and D0M5 scaffold half-on GEP.</p>
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<h3>2. Mutating aptamer binding site</h3>
<h3>2. Mutating aptamer binding site</h3>
<p>Mutating the PP7 and MS2 binding sites prevented protein scaffolding. Preventing protein scaffolding lead to the key enzyme dissociation and the decrease of enzyme local concentration. By chancing the sequence of MS2 aptamer binding site, the fluorescent light decreased. D0M3 in our project is the molecular with mutated aptamer binding site. Split GFP experiment shows that there is a significant difference between D0 an D0M3(P≦0.05, fig2.c). Camille J. Delebecque has done the same work for the H2 biosynthesis pathway.</p>
<p>Mutating the PP7 and MS2 binding sites prevented protein scaffolding. Preventing protein scaffolding lead to the key enzyme dissociation and the decrease of enzyme local concentration. By chancing the sequence of MS2 aptamer binding site, the fluorescent light decreased. D0M3 in our project is the molecular with mutated aptamer binding site. Split GFP experiment shows that there is a significant difference between D0 an D0M3(P≦0.05, fig2.c). Camille J. Delebecque has done the same work for the H2 biosynthesis pathway.</p>
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<p class="fig"><b>Fig2a.</b> MS2 and PP7 bind to the scaffold and make GFP work. </p>
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<p class="fig"><b>Fig2a.</b> MS2 and PP7 bind to the scaffold and make GFP work. </br>
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<b>Fig2b.</b> By mutating aptamer binding site, scaffolding is stop. </p>
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<p class="fig"><b>Fig2b.</b> By mutating aptamer binding site, scaffolding is stop. </p>
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<p class="fig"><b>Fig2c.</b> significant difference between D0 an D0M3</p>
<p class="fig"><b>Fig2c.</b> significant difference between D0 an D0M3</p>
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<p class="fig"><b>Fig4a.</b> Dimerization and trimerization. Protein binding site is sealed off by the scaffolds themselves. Too much scaffold molecular lend to the self regulation.</p>
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<p class="fig"><b>Fig4b.</b> Dimerization and trimerization. Protein binding site is sealed off by the scaffolds themselves. Too much scaffold molecular lend to the self regulation.</p>
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<p class="fig"><b>Fig4c.</b> Polo-scaffold be made by head-tail binding.</p>
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<p class="fig"><b>Fig4a.(Upper)</b> Dimerization and trimerization. Protein binding site is sealed off by the scaffolds themselves. Too much scaffold molecular lend to the self regulation.</br>
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<b>Fig4b.(Lower left)</b> Dimerization and trimerization. Protein binding site is sealed off by the scaffolds themselves. Too much scaffold molecular lend to the self regulation.</br>
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<b>Fig4c.(Lower right)</b> Polo-scaffold be made by head-tail binding.</p>
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<p>Several RNA scaffold mutations are constructed and characterize, but they are the tip of the iceberg. There is still plenty to do in this part. The charms of library are the selection and combination. It introduces a new concept of biobrick combination mode.</p>
<p>Several RNA scaffold mutations are constructed and characterize, but they are the tip of the iceberg. There is still plenty to do in this part. The charms of library are the selection and combination. It introduces a new concept of biobrick combination mode.</p>
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<p class="fig"><b>Fig 1.</b> pZCM (the upper one) and pZCH (the lower one)</p>
<p class="fig"><b>Fig 1.</b> pZCM (the upper one) and pZCH (the lower one)</p>
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<p class="fig"><b>Fig 4.</b> Standard curve for testing IAA concentration.</p>
<p class="fig"><b>Fig 4.</b> Standard curve for testing IAA concentration.</p>
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<p>Our experiment is on going, click <a class="parts" href="http://bis.zju.edu.cn/igem2012/project-biosyn-result.htm" target="_blank">here</a> for latest results.</p>
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                                        <h2 class="acc_trigger">07 <strong>S4: POLYSCAFFOLD</strong></h2>
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<a target="s4Frame" href="https://2012.igem.org/Team:ZJU-China/project_s4_1.htm" style="text-decoration:none">1. Summary</a><br/>
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<a target="s4Frame" href="https://2012.igem.org/Team:ZJU-China/project_s4_2.htm" style="text-decoration:none">2. Design</a><br/>
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<a target="s4Frame" href="http://bis.zju.edu.cn/igem2012/project-s4-3.htm" style="text-decoration:none">3. Results</a><br/>
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<a target="s4Frame" href="https://2012.igem.org/Team:ZJU-China/project_s4_4.htm" style="text-decoration:none">4. Future Work</a><br>
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<h2>S0: BASIC RNA SCAFFOLD</h2>
 
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<p>Contrasted to the fluorescence intensity (FI) of the E.coli which only express FA-MS2 and FB-PP7 fusion proteins, the fluorescence intensity of the E.coli with scaffold D0 was obviously increased. Thus, it was possible for us to carry out our development and reformation of RNA scaffold.</p>
 
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<p class="fig"><b>Fig.1</b> FI of Split GFPs without or with RNA scaffold. A.  BL21*(DE3) transformed with pCJDFA and pCJDFB.  B. BL21*(DE3) transformed with pCJDFA, pCJDFB and pCJDD0. The contrast of FI obviously shown that RNA scaffold D0 could bind split GFPs together, so that split GFPs could fluoresce. (Pictures were obtained with Olympus fluoview fv1000 confocal laser scanning microscope, using a 60X objective.)</p>
 
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<p>&nbsp;</p>
 
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<div class="floatC">
 
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<img src="https://static.igem.org/mediawiki/2012/6/6f/0921.png" width="500px" />
 
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</div>
 
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<p class="fig"><b>Fig.2</b>  FI/OD of different transformation groups.  There exist significant differences among three groups. And as expected, split GFPs with scaffold D0 together can fluoresce stronger than those without scaffold. </p>
 
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</br>
 
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<h3>Reference:</h3>
 
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<p class="ref">1. Thodey, K. & Smolke, C.D. Bringing It Together with RNA. Science 333, 412-413 (2011).</br>
 
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2. Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A. Organization of Intracellular Reactions with Rationally Designed RNA Assemblies. Science 333, 470-474 (2011).</p>
 
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<p>&nbsp;</p>
 
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<h2>S1: ALLOSCAFFOLD</h2>
 
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<p align="justify">&nbsp;</p>
 
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<h3>1. Scaffold</h3>
 
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<p align="justify">&nbsp;</p>
 
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<div class="floatC">
 
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<img src="https://static.igem.org/mediawiki/igem.org/5/5b/Riboscaffold_fig_12.jpg" width="500px" />
 
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</div>
 
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<p class="fig" align="justify"><b>Fig.3</b> Different RNA scaffold’s effect on split GFP showing by fluorescence microscopy. The BL21*DE3 of the E. coli were transformed with pCJDFA+pCJDFB, pCJDFA+pCJDFB + pCJDD0, and pCJDFA+pCJDFB + pZCCOV 2 (0.5 mM theophylline adding). As expected, strains without RNA scaffold did not fluoresce. Upon the existence of RNA scaffold, many of the cells emitted fluorescence indicating a substantial amount of split GFP combination is permitted because of the function of RNA scaffold. The brightfield images in the right column depict all bacterial cells. The GFP images in the left column depict bacterial cells which emitted fluorescence. </p>
 
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<p align="justify">&nbsp;</p>
 
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<div class="floatC">
 
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<img src="https://static.igem.org/mediawiki/2012/7/7f/0913.png" width="600px" />
 
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</div>
 
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<p class="fig" align="justify"><b>Fig.4</b> Biotek Synergy H1 Hybrid Reader controlled experiments. The BL21*DE3 of the E. coli were transformed with figure showing plasmids. (0.5 mM theophylline was adding in strains containing clover 2). </p>
 
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</br>
 
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<p align="justify">`luminescence \quad efficiency \quad of \quad clover 2=\frac{\frac{FI}{OD(FA+FB+clover 2)}-\frac{FI}{OD(FA+FB)}}{\frac{FI}{OD(FA+FB)}}=\frac{53425-23779}{23779}=125\%`</p>
 
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<p align="justify">&nbsp;</p>
 
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<p align="justify">`luminescence \quad efficiency \quad of \quad D0=\frac{\frac{FI}{OD(FA+FB+clover 2)}-\frac{FI}{OD(FA+FB)}}{\frac{FI}{OD(FA+FB)}}=\frac{38288-23779}{23779}=61\%`</p>
 
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<p align="justify">&nbsp;</p>
 
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<p align="justify">The original intention of our designing RNA scaffold clover 2 is to create a regulatory scaffold which can tune its conformation thus have various functions. To our surprise, clover version 2, when adding optimal Theophylline concentration 0.5mM, happens to be a more powerful scaffold which helps two halves of GFP’s combination and give out light strongly.</p>
 
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</br>
 
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<p align="justify">One possible reason is in clover version 2, distance between MS2 aptamer and PP7 aptamer is closer than in D0 (showing in 04 S1 Rboscaffold Fig.4 and Fig.6), so that when binding phage coat proteins, FA and FB on clover version 2 were set closer than on D0. We submit the inference that when RNA scaffold binds enzymes, clover version 2 draws two enzymes nearer than D0 thus has more ability to accelerate the enzymatic reaction.</p>
 
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</br>
 
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<h3>2. Regulate and control by Theophylline</h3>
 
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<p align="justify">When the concentration of Theophylline is in the range from 0mM to 0.5mM, the concentration of Theophylline and the resulting fluorescence intensity are directly proportional. </p>
 
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</br>
 
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<p align="justify">Theophylline concentration beyond certain extent will be hazardous to cells and how it affects cells depends on strain type. The study by NYMU Taipei 2010 alerted adding more than 4mM of Theophylline would cause E. coli to die. In our experiments, we find that after adding more than 0.5mM, the Theophylline spectrum curve would be invalid. As a result, we pick up data with concentrations below 0.5mM to analyze as the E. coli cell would be unstable or the regulation of the Theophylline aptamer would not be accurate. </p>
 
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</br>
 
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<img src="https://static.igem.org/mediawiki/igem.org/2/20/Screen_Shot_2012-09-26_at_%E4%B8%8B%E5%8D%885.27.52.png" width="700px" />
 
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<p class="fig" align="justify"><b>Fig.5</b> origin data of clover 2 regulatory tests. First line of each form is different treatments of Theophylline concentration and data in table cells are fluorescence intensity/ OD.</p>
 
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<p>&nbsp;</p>
 
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<div class="floatC">
 
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<img src="https://static.igem.org/mediawiki/2012/e/ed/Final_clover2.png" width="500px" />
 
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</div>
 
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<p class="fig" align="justify"><b>Fig.6</b> 7 tests of fluorescence/ OD change over theophylline concentration. There’s evident positive correlation in between.</p>
 
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<p>&nbsp;</p>
 
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<p align="justify">Then we build several SAS models to analyze data with SAS software GLM procedure between 0-0.5mM Theophylline concentrations of treatments, choosing” clover version 2: different treatments versus blocks” test 5-7 to run a SAS model.</p>
 
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</br>
 
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<p align="justify">ANOVA result P-value shows that Theophylline concentrations have significant impact on fluorescence intensity of clover version 2 and almost no impact on D0. That is to say, our designed RNA scaffold clover version 2 can be regulated and controlled by Theophylline within 0-0.5mM not for random errors or common phenomenon in RNA scaffolds.</p>
 
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</br>
 
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<p align="justify">If you want more details about SAS source programs and software computational results, please click here <a class="parts" href="https://2012.igem.org/Team:ZJU-China/sourcecode1.htm" target="_blank">[code]</a>. </p>
 
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</div>
 
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Latest revision as of 03:12, 27 October 2012

PROJECT

01 ABSTRACT

02 BACKGROUND

03 S0: BASIC RNA SCAFFOLD

04 S1: ALLOSCAFFOLD

05 S2: SCAFFOLD LIBRARY

06 S3: BIOSYNTHESIS OF IAA

07 S4: POLYSCAFFOLD

08 PARTS

09 PERSPECTIVES