http://2014.igem.org/wiki/index.php?title=Special:Contributions/Soipan&feed=atom&limit=50&target=Soipan&year=&month=2014.igem.org - User contributions [en]2024-03-28T21:37:12ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:SCUT/Project/Other_WorkTeam:SCUT/Project/Other Work2014-10-18T02:44:01Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation"><img src="https://static.igem.org/mediawiki/2014/4/44/Project2-01.png"></a><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation" id="special" title="A Link"><p>CO<sub>2</sub> Fixation>>></p></a><br />
<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation" id="special" title="A Link"><p>n-Butanol Prod>>></p></a><br />
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<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
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<p onclick="scroll_1()">TQPA</p><br />
<p onclick="scroll_2()">Leading Peptides</p><br />
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<p class="atop"><br />
<span>Testing and Quantification of Promoter Activities</span><br />
</p><br />
<p><span class="xiaobiaoti">Overview</span><br/><br/><br />
<br />
This is an additional part of our project, designed to testing and quantify promoter activities for having moderate expression level of target protein, or more detailedly saying, for knowing if TEF2 and TDH3 promoter work and for better control of induction of Gal1 promoter, the only inducible promoter used in our project. We employed GFPmut3b ( BBa_K294055 ), one of the most widely used fluorescence protein, as a reporter. After making a comparison between FCM and ELIASA, we chose the latter one for quantification of fluorescence since it was more convenient to manipulate and was accurate enough for comparing promoter activities. Considering that the fermentation process will span a period of time from several hours to several days, we did not have timed induction of Gal1 promoter included in quantification process. <br />
</p><br />
<p><br />
<span class="xiaobiaoti">Design and Result</span><br/><br/><br />
With employing GFPmut3b (<a href="http://parts.igem.org/Part:BBa_K294055:Design">BBa_K294055</a>) as a reporter protein, we designed three similar devices only differing in promoters as shown below.</p><br />
<p><b>Promoter Testing</b><br/><br />
Plasmids carrying the above-mentioned devices were transformed into competent S.cerevisiae cells. Transformed cells were plated on 2% agar in media A (yeast nitrogen base, MET, LYS, HIS, URA), complemented with 2% glucose and cultured at 30℃ for 48h. The colonies were then inoculated in the liquid media A complemented with 2% glucose and were grown at 30℃ overnight. For those contained TEF2 or TDH3 promoter, culture was stopped and samples were taken out for testing. For those contained Gal1 promoter, glucose was removed from media with washing cells by PBS till they reached the exponential phase. Cells were then pelleted and diluted and cultured on liquid medium containing galactose concentration of 2% w/v overnight. Samples were then taken out for testing. The result is shown below.</p><br />
<img src="https://static.igem.org/mediawiki/parts/b/bd/GAL1.jpg"><br />
<img src="https://static.igem.org/mediawiki/parts/4/46/TDH3.jpg"><br />
<img src="https://static.igem.org/mediawiki/parts/1/1b/TEF2.jpg"><br />
<p class="m-zhujie"><b>Figure 1 丨 The results show that all the promoters work.</b></p><br />
<p><br />
<b>Galactose Dose Response of Gal1 Promoter</b><br />
<br/><br />
Plasmid carrying the device containing Gal1 promoter was transformed into competent S.cerevisiae cells. Transformed cells were plated on 2% agar in media A (yeast nitrogen base, MET, LYS, HIS, URA), complemented with 2% glucose and cultured at 30℃ for 48h. The colonies were then inoculated in the liquid media A complemented with 2% glucose and were grown at 30℃. Culture was stopped and glucose was removed from media with washing cells by PBS till they reached the exponential phase. Cells were then pelleted and diluted and cultured on liquid medium containing galactose concentrations between 0.25% and 2% w/v overnight. Samples were taken out for quantification.<br />
</p><p><br />
In quantification process, concentrations of liquid media containing cells were measured at λ600nm and then liquid media were removed and cells were washed twice and refolded to a OD600 = 1.0 concentration by Buffer ( 0.05 M NaH2PO4, 0.1 M NaCl, 0.5 M Imidazole ) for ELIASA analysis. Fluorescence intensity of the refolded cells were then measured by ELIASA (Tecan, Infinite M200 and i-Control)) with an excitation wavelength of 488 nm (the suggested one is 501 nm), and an emission filter of 520 nm (the suggested one is 511nm) to quantify fluorescence intensities. The result is shown below.<br />
</p><br />
<p ><br />
<img src="https://static.igem.org/mediawiki/parts/b/bb/Dose.jpg"><br />
</p><br />
<p class="zhujie"><b>Figure 2 丨 Dose responsiveness of the Gal1 promoter to galactose induction</b></p><br />
<p><br />
The graph above summarises the ELIASA data, and shows that the intensity of GFP expressing cells induced by low or high dose of galactose are higher then those induced by the middle doses. This is similar to the result given by iGEM 2010 team, Aberdeen_Scotland. We therefore plan to set more experiment groups between these doses to figure out what happened.</p><br />
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<span>Leading Peptides for Future Plan</span><br />
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<p>What we plan to do next is to find the other pathways in the yeast and make it highly effective by taking advantages of the micro environments. But frist of all, how to make the protein import into its own station and work smoothly? Fortunately, we find some leading peptides to be our “guides”. As we know, different locations need different “guides”. This summer, we have constructed 6 leading peptides successfully in the yeast. In other words, we have 6 another possibility to take advantages of the micro environments.</br>We chose those 8 membrane from a list of about ten candidates based on the following factor:</br>1.It can be easily targeted in yeast.</br>2.Less reaction over it is not allowed.</br>3.Visible distinction from others.</br>So far,we have found the following leading peptides.<br />
</p><br />
<p><br />
<span><br />
1.Erv25p<br />
</span><br />
<br/><br />
We characterized a novel protein termed Erv25p that have benn discovered on ER-derived transport vesicles, and it is required for efficient ER to golgi transport. Erv25p is a single membrane spanning segment derived from yeast, and a 12-amino acid N-terminal sequence exposed to the cytoplasm.</br><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/5/59/Erv25p-Y.jpg"><br />
</br><br />
</p><br />
<p class="zhujie"><br />
Figure 3<b>丨Direct targeting of proteins to ER丨</b>The gene Erv25p(<a href="http://parts.igem.org/Part:BBa_K1462940">BBa_K1462940 </a> ) was obtained from S. Cerevisiae genome by PCR technique.BFP was fused to its C-terminus as reporter.The biobrick is constructed as follow to verify this leading peptide.</br><br />
</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/5/5a/BB-Erv25p.png"><br />
</p><br />
<br />
<p><br />
2.CTS1-1</span></br>A N-terminal 12-amino acid serve as a typical cleavable signal sequence. The targeting sequence derived from yeast can be located at cytoderm.</br><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/f/f0/Cts-Y.jpg"><br />
</br><br />
</p><br />
<p class="zhujie"><br />
<b>Figure 4 丨 Direct targeting of proteins to cytoderm丨</b>We got this short targeting sequence(<a href="http://parts.igem.org/Part:BBa_K1462930">BBa_K1462930</a>) by gene synthesis .GFP was fused to its C-terminus as reporter. The biobrick is constructed as follow to verify this leading peptide.<br />
</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/e/e0/BB-CTS1-1.png"><br />
</p><br />
<br />
<p><br />
3.CIIC</span></br><br />
It’s a 12-amino acid C-terminal sequence separated from the Ras protein,which is the leading peptide of Plasma Membrane.<br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/e/e3/CTS1-1--Y.jpg"><br />
</br><br />
</p><br />
<p class="zhujie"><br />
<b>Figure 5 丨Direct targeting of proteins to Plasma Membrane丨</b>CIIC(<a href="http://parts.igem.org/Part:BBa_K1462850">BBa_K1462850</a>) target to We got this short targeting sequence by gene synthesis .CFP was fused to its N-terminus as reporter.The biobrick is constructed as follow to verify this leading peptide. iGEM12_Berkeley also use a same sequence.<a href="http://parts.igem.org/Part:BBa_K900005">BBa_K900005</a><br />
</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/2/27/BB-CIIC.png"><br />
</p><br />
<br />
<p><br />
4.H2A2</span></br><br />
The localizing protein(H2A2)(<a href="http://parts.igem.org/Part:BBa_K1462870">BBa_K1462870</a>) is the major structural protein of chromosomes. And the C-terminal protein fusion to it will be located at nucleus.</br>The gene H2A2 was obtained from S. Cerevisiae genome by PCR technique.CFP was fused to its C-terminus as reporter.<br />
The biobrick is constructed as follow to verify this leading peptide.<br />
</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/9/94/BB-H2A2.png"><br />
</p><br />
<br />
<p><br />
5.ZRC1</span></br><br />
The localizing protein(ZRC1)(<a href="http://parts.igem.org/Part:BBa_K1462860">BBa_K1462860</a>)is a protein of vacuolar membrane zinc transporter.And a C-terminal protein fusion to it will be located at vacuolar membrance.</br>The gene ZRC1 was obtained from S. Cerevisiae genome .YFP was fused to its C-terminus as reporter.<br />
The biobrick is constructed as follow to verify this leading peptide.</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/3/3d/B-ZRC1.png"><br />
</p><br />
<br />
<p><br />
6.PTS</span></br><br />
There is a mechanism about cargo translocation in the peroxisomes, the matrix proteins are posttranslationally targeted to peroxisomes from the cytosol by peroxisomal targeting signals (PTSs). These signals include the predominantly used PTS1(<a href="http://parts.igem.org/Part:BBa_K1462910">BBa_K1462910</a>) and the less prevalent PTS2, which are recognized by the soluble import receptors PEX5 and PEX7, respectively.<br />
</br> We obtained this nine-bp-leading peptide by gene synthesis .GFP was fused to its C-terminus as reporter. The biobrick is constructed as follow to verify this leading peptide.</p><br />
<p class="image"><br />
<img src="https://static.igem.org/mediawiki/parts/b/b2/BB-PTS1.png"><br />
</p><br />
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<p class="atop"><br />
<span>References</span><br />
<p>[1] Michael J. KurandaS and Phillips W. Robbins : <b>Chitinase Is Required for Cell Separation during Growtho f<br />
Saccharomyces cerevisiae".</b> The Journal of Biological Chemistry 1991 by The American Society for Biochemistry and Molecular Biology, Inc.<br />
</p><br />
<p>[2] William J. Belden and Charles Barlowe J. Biol. Chem.: <b>Reticulum to Golgi Transport That Is Required for Efficient Endoplasmic<br />
Vesicles, Forms a Complex with Emp24p Erv25p, a Component of COPII-coated.</b> The Journal of Biological Chemistry 1996, 271:26939-26946. <br />
doi: 10.1074/jbc.271.43.26939<br />
</p><br />
<p>[3] Jennifer J. Smith and John D. Aitchison: <b>Peroxisomes take shape</b> NATURE REVIEWS 98109-5219, USA. <br />
doi: 10.1074/jbc.271.43.26939<br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_FixationTeam:SCUT/Project/System Construction/Co2 Fixation2014-10-18T02:41:53Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/CO<sub>2</sub> Fixation" id="special" title="A link"><p>CO<sub>2</sub> Fixation>>></p></a><br />
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<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Design</p><br />
<p onclick="scroll_3()">Construction</p><br />
<p onclick="scroll_4()">Leading Peptide Test</p><br />
<p onclick="scroll_5()">References</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/n-Butanol Prod" id="special" title="A link"><p>n-Butanol Prod>>></p></a><br />
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<p>Introduction</p><br />
<p>Design</p><br />
<p>Construction</p><br />
<p>Localization Test</p><br />
<p>References</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Analysis_and_Discussion"><img src="https://static.igem.org/mediawiki/2014/f/fe/Project3-01.png"></a><br />
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<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
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<p>Outline</p><br />
<p>Reference</p><br />
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<span>Introduction</span><br />
</p><br />
<p>The components of the enzyme machinery anchored onto the outer mitochondrial membrane were selected within the known endogenous metabolic enzymes of S. cerevisiae and other exogenous enzymes, considering the high availability of ATP and CO<sub>2</sub> around mitochodria. The selected enzymes were PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) that working sequentially improve the carbon sequestration of the yeast,or in other words, increase the yield of pyruvate thereby increasing butanol production. To maximize the use of ATP and CO<sub>2</sub>, a selected leading peptide (Tom22) was used to anchor three exogenous scaffold proteins (GBD, SH3 and PDZ) able to bind the above-mentioned enzymes within a limited distance from the membrane.<br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/parts/0/08/Rubisco-cut.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 1 | The overview of our project.</b> </p><br />
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</p><br />
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<span class="xiaobiaoti">PRK</span><br />
<br />
<p><br />
A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:<br />
<br/><br />
<b>ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate</b><br />
<br/><br/><br />
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.<br />
<br/><br/><br />
The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway. <br />
<br/><br/><br />
</p><br />
<span class="xiaobiaoti">RuBisCo</span><br />
<p><br />
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. <br />
<br/><br />
<b>Ribulose 1,5-bisphosphate + CO<sub>2</sub> → 2x 3-phosphoglycerate</b><br />
<br/><br/><br />
In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O<sub>2</sub>) instead of carbon dioxide (CO<sub>2</sub>). Thus, something must be taken to increase concentration of the substrate of interest (CO<sub>2</sub>)to bring a high yield. <br />
<br/><br/><br />
The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway<br />
</p><br />
<span class="xiaobiaoti">CA</span><br />
<br />
<p>Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide<br />
and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes.<br />
The reaction catalyzed by carbonic anhydrase is:<br />
<br/><br />
<b>H<sub>2</sub>CO<sub>3</sub> → CO<sub>2</sub> + H<sub>2</sub>O</b><br />
<br/><br/><br />
<br />
The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.<br />
<br/><br/><br />
The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.<br />
</p><br />
<br />
<span class="xiaobiaoti">Chaperons</span><br />
<p>It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol. <br />
<br/><br/><img src="https://static.igem.org/mediawiki/parts/1/1e/Molecular_chaperone.jpg"><br />
<p style="margin-top:10px;"><b><br />
Figure 2 | The schematic of mechanism of the molecular chaperons GroES and GroEL.</b> Firstly, an ATP binds with GroEL and the ATP-GroEL immediately captures the other unit GroES and there comes the GroEL-GroES-ATP complex.With the help of the complex, the unfolded RuBisCo protein can then properly fold and make a difference in our project.<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Scaffold Protein</span><br />
<p><br />
Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.<br />
<br/><br/><br />
Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/1a/TOM22.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 3 | </b>As shown in the picture, we construct the scaffold protein fusing with the Tom22 which is the member of the Tom family. Tom family is the protein complex that helps the protein including leading peptide enter into the mitochondria. We utilized the embed protein Tom22 to make our scaffold protein (the scaffold protein fuse with the Tom22).<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Leading Peptides</span><br />
<p><br />
In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.<br />
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<span>Design</span><br />
</p><br />
<p><br />
Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.<br />
</p><br />
<p><br />
We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.<br />
</p><br />
<p><br />
The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.<br />
</p><br />
<p><br />
It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in <a href="https://2014.igem.org/Team:SCUT/Project/Other_Work">Quantification of Promoter Activities</a>).<br />
</p><br />
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<span>Construction</span><br />
</p><br />
<p>The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.<br />
We combine not only our enzymes PRK, RuBisCo and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBisCo and CA</p><p><br />
These are constructions of our pathway and scaffold protein.<br />
<img src="https://static.igem.org/mediawiki/parts/d/d8/Plasmid-firedfish.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 4 |</b> We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA were built up in the plasmid Yeplac181. Moreover, the FP + ligand was also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid YEp352.<br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/19/Bigna.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Fiugre 5 | </b>The SCUT A is the pathway that the RuBisCo contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.<br />
</p><img src="https://static.igem.org/mediawiki/parts/0/0b/Fermentation.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 6 | At the pre-experiment</b>, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other(group Ⅱ) consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis.<br />
As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells(group 0). With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_4"><br />
<p class="atop"><br />
<span>Leading Peptide Testing</span><br />
</p><br />
<p>We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.<br />
</p><p><br />
<img src="https://static.igem.org/mediawiki/parts/8/85/Bgy.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 7 | </b>This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial <br />
membrane.<br />
</p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/2BGY.jpg"></p><br />
<p style="margin-top:10px;"><br />
<b>Figure 8 | </b> The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.<br />
</p><br />
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</p><br />
</p><br />
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<div class="mainbody" id="label_5"><br />
<p class="atop"><br />
<span>References</span><br />
</p><br />
<p>[1] Hillel K. Brandes‡, Fred C. Hartman§¶, Tse-Yuan S. Lu§, et al. : <b>Efficient Expression of the Gene for Spinach Phosphoribulokinase in Pichia pastoris and Utilization of the Recombinant Enzyme to Explore the Role of Regulatory Cysteinyl Residues by Site-directed Mutagenesis*.</b> The Journal of Biological Chemistry 1996 Vol. 271, No. 11, Issue of March 15, pp. 6490–6496.<br />
</p><p><br />
[2] F. Robert Tabita, Sriram Satagopan, Thomas E. Hanson, et al. : <b>Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.</b> Journal of Experimental Botany 2008 Vol. 59, No. 7, pp. 1515–1524.<br />
</p><p><br />
[3] Christopher R. Somerville and Shauna C. Somerville: <b>Cloning and Expression of the Rhodospirillum rubrum Ribulosebisphosphate Carboxylase Gene in E. coil.</b> Mol Gen Genet 1984 193:214-219<br />
</p><p><br />
[4] Víctor Guadalupe-Medina, H Wouter Wisselink, Marijke AH Luttik, et al. : <b>Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast.</b> Biotechnology for Biofuels 2013 6:125.<br />
</p><p><br />
[5] Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, et al. : <b>Modularity of a carbon-fixing protein organelle.</b> PNAS 2012 vol. 109 , no. 2: 478-483.<br />
</p><p><br />
[6] Jian Qiu, Lena-Sophie Wenz, Ralf M. Zerbes, et al. : <b>Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation.</b> Cell 2013 154, 596–608.<br />
</p><p><br />
[7] Birgitta M. GEIER', IIermann SCHAGGER', Claus ORTWEIN", et al. : <b>Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated.</b> Eur. J. Biochem. 227, 296-302 (1995). Eur. J. Biochem 1995 227, 296-302.<br />
</p><p><br />
[8] MA Jun, SUN Fei: <b>Translocation of Mitochondrial Proteins.</b> ACTA BIOPHYSICA SINICA 2010 Vol.26 No.10: 880-893<br />
</p><p><br />
[9] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al. : <b>Synthetic protein scaffolds provide modular control.</b> NATURE BIOTECHNOLOGY 2009 Vol. 27 No. 8<br />
</p><p><br />
[10] Matthew C. Good, Jesse G. Zalatan, Wendell A. Lim† : <b>Scaffold Proteins: Hubs for Controlling the Flow of Cellular.</b> Science 2011 332, 680.<br />
</p><p><br />
[11] Tae SeokMoona, JohnE.Dueber, EricShiue, et al. : <b>Use of modular, synthetic scaffolds forim proved production of glucaricacid in engineered E. coli over metabolic flux.</b> Metabolic Engineering 2010 12 298–305. </p><br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/OverviewTeam:SCUT/Project/Overview2014-10-18T02:41:26Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<div class="navihead navihead2"><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation" id="special" title="A Link"><p>CO<sub>2</sub> Fixation>>></p></a><br />
<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation" id="special" title="A Link"><p>n-Butanol Prod>>></p></a><br />
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<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>TQPA</p><br />
<p>Leading Peptides</p><br />
</div><br />
</div><br />
<br />
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<div class="mainbody mainbody1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p><br />
A cellular compartment is an intracellular space enclosed by a single or double lipid layer membrane that separates specific molecules and molecular machineries from the cytosol. Different intracellular pH, different enzyme systems and other substances are isolated within membrane-bound compartments. Due to compartmentalization and positional assembly, a cell can perform different metabolic activities at the same time. This generates specific micro-environments that regulate multistep processes. This project aimed to develop methodologies that allow a better control over complex synthetic reactions. <br />
</p><p><br />
Mitochondrion is a membrane-enclosed organelle that generates the energy currency of the cell, i.e. ATP, through cellular respiration. In the tricarboxylic acid (TCA) cycle, each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane into the matrix where it is oxidized and combined with coenzyme A to form CO<sub>2</sub>, acetyl-CoA, and NADH. As a consequence, the environment in the mitochondrial matrix has a lower oxygen concentration, a higher pH and a more reducing redox potential than the cytoplasm. This is beneficial to the activity of several enzymes that need reduced cofactors which are synthesized exclusively in mitochondria. <br />
</p><p><br />
<p><br />
In order to take advantage of the particular features of the mitochondrial environment and increase the effective concentration of each component of an n-butanol pathway, we transferred the complete n-butanol production pathway into the yeast mitochondrial matrix using several leading peptides. Furthermore, a parallel assembly of three carbon sequestration enzymes were organized onto the outer membrane of mitochondria with a scaffold protein. With this machinery, the waste byproduct CO<sub>2</sub> released by the TCA cycle was recycled to produce pyruvate, thereby increasing the substrate supply for the production of n-butanol (Fig. 3).<br />
</p><p><br />
<br />
<img style="width:700px;" src="https://static.igem.org/mediawiki/parts/d/d1/Project2.jpg"><br />
<p class="zhujie"> Fig. 1 The engineered pathway. The complete n-butanol pathway was targeted into the &nbsp;&nbsp;&nbsp;&nbsp;mitochondrial matrix. The carbon sequestration enzymes were anchored onto the outer membrane of mitochondria to recycle the CO<sub>2</sub> released through TCA. The darkness of the green indicates concentration of CO<sub>2</sub>: the darkest green marks the highest value.<br />
</p><br />
<br />
</p><p><br />
Compartmentalization of metabolic pathways into a controlled organelle has been proved to increase the target production effectively. Here, the new concept of PAN-compartmentalization is introduced, which considers both the inner side and the outer side of the compartment as the target. The core principle of pan-compartmentalization is to achieve the higher enzymes concentration, to recycle byproducts and to increase the availability of substrates, in order to maximize the carbon recovery and the efficient use of coenzymes. Simple word is good understanding and make the best of the environment in/out of the specific compartment.<br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/Tips_for_other_teamsTeam:SCUT/Model/Tips for other teams2014-10-18T02:41:10Z<p>Soipan: </p>
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<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol part</p><br />
</div><br />
<div class="navihead navihead2"><a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
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<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Complete Network</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
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<div class="navibody navibody3"><br />
<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
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<p onclick="scroll_1()">Outline</p><br />
<p onclick="scroll_2()">Reference</p><br />
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<p class="atop"><br />
<span>Outline</span><br />
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<p class="first"><br />
In the process of searching for data, we have met a lot of difficulties. On the one hand, the parameters for the pathway we want are difficult to find. On the other hand, even when we find the paper we need, the data on the paper is too many to figure out what is we exactly want. However, after numerous times of searching, we find that there exists a wonderful database, the <span>BioModel Database </span><span onclick="scroll_a()" id="yin">[1]</span>. It is a useful library for those who want to find the data and models for biology. Besides, the data is showed in a brief way, which is easy to read and use. So we highly recommend other iGEM teams to browse this database when you need to find the models for your project.<br/><br />
The other powerful tool we want to share with you is the<span> JWS online</span><span onclick="scroll_a()" id="yin">[2]</span>, an online lab for the models that have already uploaded to it. It can give you the results you want in a few seconds, which can help you immediately grasp the features of the model. We also use the results on it to exam the correctness of our code by finding if there are any differences between our results and theirs. For example, we use the results of the online lab to compare with our results of glycolysis subsystem. Since the glycolysis model is the most complex one, it is difficult to exam the code row by row. With the help of the<span> online lab</span>, we can easily test whether there are bugs of our code, which has saved us a lot of time. The only limitation of<span> JWS online</span> is that it cannot combine different models in it to construct a more powerful system. However, it is still powerful, isn’t it?<br />
</p><br />
<p id="tip"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/Tip1.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/48/Tip2.png"><br/><br />
Figure 1 the figures of our code and the corresponding online model. <br />
(a) is part of the results of our code, (b) is the corresponding figure.<br />
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<div class="mainbody mainbody2" id="label_2"><br />
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<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1] <a href="http://www.ebi.ac.uk/biomodels-main/">http://www.ebi.ac.uk/biomodels-main/</a>, BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic <br/><br />
[2] <a href="http://jjj.mib.ac.uk/database/">http://jjj.mib.ac.uk/database/</a>, JWS online<br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T02:40:36Z<p>Soipan: </p>
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<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
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<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
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<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
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<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
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<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
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<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
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<span>Simulation</span><br />
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<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
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<p><br />
The rate expression is defined as<br />
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<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
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<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
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<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:28px;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/Rubisco_simulationTeam:SCUT/Model/Rubisco simulation2014-10-18T02:40:17Z<p>Soipan: </p>
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<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol part</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Individual part</p><br />
<p onclick="scroll_3()">Complete Network</p><br />
<p onclick="scroll_4()">The function of RuBisCo</p><br />
<p onclick="scroll_5()">Scaffold</p><br />
<p onclick="scroll_6()">Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3"><br />
<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
The <span>“bottom up” method </span>is a helpful tool to solve our problem. By applying it, we make the original problem simple, that is, dividing the original network into three parts, and then merging them together to build a complete system. With the guide of this principle, we choose the<span> ODEs </span>to model our individual part. The reason why we use the <span>ODEs</span> consists of two parts. One is that they are feasible to combine together to construct a whole network. The ODEs are just like components, which have interfaces to communicate with others easily. Another one is that we can simply <span>sweep the parameters</span> to simulate the changed experiment conditions.<br />
</p><br />
<p><br />
The steps for our work are as follows:<br />
</p><br />
<p><br />
1. Since the pathway consists of three parts, which are PPP(pentose phosphate pathway),<span> glycolysis </span>and<span> part of Calvin Cycle</span>, we firstly find the parameters and model them individually.<br />
</p><br />
<p><br />
2. After making sure the correctness of our individual parts of modeling, we combine them together to figure out the performance of the pathway.<br />
</p><br />
<p><br />
3. By comparing the difference of the production of ethanol between with and without the part of Calvin Cycle, we present<span> the function of RuBisCo</span>. <br />
</p><br />
<p><br />
4. By using parameter sweep, we find out the <span>optimal reaction rate ratio </span>of the reactions involved in the scaffold, thus proving the function of scaffold and<span> showing the internal limitation of the pathway</span>.<br />
</p><br />
</div><br />
<br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Individual part</span><br />
</p><br />
<p class="first"><br />
The ODEs is mainly based on <span>Michaelis-Menten equation</span>. However, the equation we used is an <span>improved version</span> of Michaelis-Menten equation, that is, by introducing more parameters to indicate the factors influencing the reaction. Next, let us show them individually.<br />
</p><br />
<p><br />
<span>1. PPP(pentose phosphate pathway)</span><br />
</p><br />
<p style="margin-top:-30px;"><br />
According to what we have investigated <span onclick="scroll_a()" id="yin">[1]</span>, we build the <span>PPP</span> subsystem. The patterns of the equations are similar. In order to make our presentation brief, we only introduce one of the equations and recommend you to visit the<span> BioModel Database</span> <span onclick="scroll_a()" id="yin">[2]</span> for detail. (For more information, please see the “<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams">Tips for other teams</a>” part). Take GND reaction as an example, the equation of GND reaction rate and the parameters are showed as follows:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e0/Equ_1_%281%29.png" ><br />
</p><br />
<p><br />
The result are showed in figure 1:<br />
</p><br />
<p style="margin-top:-30px;" id="f1"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6f/A.PNG" class="diyi"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/B.PNG" ><br />
<img src="https://static.igem.org/mediawiki/2014/9/99/C.PNG"><br/><br />
Figure 1 The result of PPP subsystem<br />
</p><br />
<p><br />
<span>2. Glycolysis</span><br />
</p><br />
<p style="margin-top:-30px;"><br />
The <span>glycolysis</span> in yeast is the most complex model<span onclick="scroll_a()" id="yin">[3]</span> since it consists of numerous reactions and different kinds of mechanisms<span onclick="scroll_a()" id="yin">[4]</span>. Just as what we have done in the PPP part, that is, to make the presentation brief, we will show the representation of each kind of mechanism and suggest you to browse the<span> BioModel Database</span><span onclick="scroll_a()" id="yin">[2]</span> for detail.<br />
</p><br />
<p style="margin-top:-30px;"><br />
For the reactions PGI, PGM and ENO, they can be modeled by using one substrate, one product reversible Michaelis-Menten kinetics:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d9/Equ_1_%282%29.png"><br />
</p><br />
<p><br />
where a and p represent the concentrations of the corresponding substrate and product respectively, R is the mass-action ratio, p/a, Keq is the equilibrium constant, peq/aeq. Ka and Kp are the Michaelis-Menten constants for a and p.<br/><br />
For the reactions HK, GraPDH, PGK and PYK, we use reversible Michaelis-Menten kinetics for two noncompeting substrate-product couples to model them:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%283%29.png"><br />
</p><br />
<p><br />
where a and b represent the concentrations of the substrates , p and q represent the concentrations of the products.<br />
For the transport of glucose:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d8/Equ_1_%284%29.png"><br />
</p><br />
<p><br />
where Ki is the interactive constant, [Glcout] and [Glcin] are the concentrations of extracellular and intracellular glucose respectively.<br />
For ALD, which follows an ordered uni-bi mechanism, the equation is:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/Equ_1_%285%29.png"><br />
</p><br />
<p><br />
where a represent the substrate [F16bP], p and q represent the products [GPP] and [GraP] respectively.<br/><br />
For PDC, irreversible hill kinetics are used to describe the reaction rate:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c3/Equ_1_%286%29.png"><br />
</p><br />
<p><br />
For ADH, it follows ordered bi-bi kinetics<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/Equ_1_%287%29.png"><br />
</p><br />
<p><br />
For ATPase, it is the simplest one:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/96/Equ_1_%288%29.png"><br />
</p><br />
<p><br />
For PFK, it is the most complex one:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%289%29.png"><br />
</p><br />
<p><br />
with:<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2b/Equ_1_%2810%29.png"><br />
</p><br />
<p><br />
After finding out the equations and parameters, we run the program and get the following result( figure 2).<br/><br />
</p><br />
<p id="f2"><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/F2a.PNG" class="diyi"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/F2b.PNG" class="diyi"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/F2c.PNG"><br />
<img src="https://static.igem.org/mediawiki/2014/4/46/F2d.PNG"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/F2e.PNG"><br />
<img src="https://static.igem.org/mediawiki/2014/7/7a/F2f.PNG"><br />
<img src="https://static.igem.org/mediawiki/2014/1/15/F2g.PNG"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2e/F2h.PNG"><br />
<span style="margin-left:200px;">Figure 2 The result of glycolysis subsystem</span><br />
</p><br />
<p><br />
<span>3. Part of Calvin Cycle</span><br />
</p><br />
<p style="margin-top:-30px;"><br />
This part is <span>the most significant part</span> of our network since it is the part we introduce into the yeast to <span>improve the production of ethanol</span>. It involves two reactions, whose kinetic equations follow the classic <span>Michaelis-Menten kinetics </span><span onclick="scroll_a()" id="yin">[5]</span>.<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8f/Equ_1_%2812%29.png"><br />
</p><br />
<p><br />
This part is so small that we didn’t run it individually and we will see its function in the Complete network and The function of RuBisCo.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Complete network</span><br />
</p><br />
<p class="first"><br />
After we make sure the correctness of each part, that is, by comparing the running results with the paper results or the online math lab running results<span onclick="scroll_a()" id="yin"> [6] </span>to see if our results are consistent with them.(For more information, see the part "<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams">Tips for other teams</a>"), we combine three parts together to construct our complete network. The results of our complete network are showed in figure 3.<br />
</p><br />
<p id="f3"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/F3a.png" class="diyi"><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/F3b.png" class="diyi"><br />
<img src="https://static.igem.org/mediawiki/2014/8/84/F3c.png"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/F3d.png"><br />
<img src="https://static.igem.org/mediawiki/2014/8/86/F3e.png"><br />
<img src="https://static.igem.org/mediawiki/2014/9/92/F3f.png"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b7/F3g.png"><br />
<img src="https://static.igem.org/mediawiki/2014/0/0b/F3h.png"><br />
<img src="https://static.igem.org/mediawiki/2014/6/60/F3i.png"><br />
<img src="https://static.igem.org/mediawiki/2014/7/79/F3j.png"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9d/F3k.png"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/F3l.png"><br />
<span style="margin-left:200px;">Figure 3 The results of the whole system</span><br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>The function of RuBisCo</span><br />
</p><br />
<p class="first"><br />
In order to test <span>the function of RuBisCo</span>, that is, the enzyme we have introduced into the yeast, we <span>compare the production of ethanol</span> of two different networks, one is with the RuBisCo while the other is the original one. In fact, the network with RuBisCo is the network we have presented in the Complete work, and the original one is the network without part of Calvin Cycle. We <span>construct two different ODEs system</span> to see the change of production of ethanol. The comparison result shows <span>the function of RuBisCo</span>, which is<span> improving the production of ethanol</span>. From figure 4, we can see that with the introduction of RuBisCo, the ethanol yield has been increased by 8.3868%. (This data is calculated in MATLAB and displayed on the command window)<br/><br />
</p><br />
<p id="f4-5"><br />
<img src="https://static.igem.org/mediawiki/2014/4/45/Figure_4.PNG" class="f3-4"><br/><br />
Figure 4 the comparison result of the networks with and without RuBisCo<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Scaffold</span><br />
</p><br />
<p class="first"><br />
<br />
In addition to introduce RuBisCo into yeast, we also assemble that part on the scaffold for it can increase the effective concentrations of metabolic intermediates <span onclick="scroll_a()" id="yin">[7]</span>. As a result, we wonder <span>the optimal reaction rate ratio</span>, that is, the best multiples of the original reaction rate, for our network.<br/><br />
To find out the optimal ratio, we <span>sweep the parameters</span> of the reactions that assembling on the scaffold to see the change of production of ethanol. For all of the parameters to be discussed, vmax is the most important one. Consequently, we choose the<span> vmax </span>to sweep. We plot the production-reaction rate ratio figure to make the change more straightforward. According to the result (figure 5), we can make the conclusion that with the improvement of reaction rate ration, the product become more and more large. However, when the ratio has become 20 multiples of the original one, which is with RuBisCo but without scaffold, the change of production become small and can be ignored. So the <span>optimal reaction rate ratio<span> is approximately 20 multiples. And at that situation, the production of ethanol has been increased by about 5% when comparing to the one with RuBisCo but without scaffold. <br/><br />
Maybe you will find the trend is strange and ask, why it isn’t showed like that with the increase of reaction rate, the product also increase without limitation. To explain such strange thing, we should firstly show the reason why RuBisCo can increase the production of ethanol. It is because the RuBisCo pathway is a<span> competitive pathway </span>with the fermentation of glycerol <span onclick="scroll_a()" id="yin">[8]</span>. The glycerol is one of the by-products of the production of ethanol, and with the introduction of RuBisCo, we can <span>redistribute the nutrients to the direction we want</span>, which is improving the production of ethanol. And by using scaffold, we can<span> enhance the performance of RuBisCo</span>. However, the fermentation of glycerol is <span>limited</span>, when we <span>achieve the better situation</span>, that is, making the production of glycerol become zero, we cannot enhance the performance of RuBisCo anymore.<br />
</p><br />
<p id="f4-5"><br />
<img src="https://static.igem.org/mediawiki/2014/a/a6/Figure_5.PNG"><br/><br />
Figure 5 The relationship of the product and the reaction rate ratio<br />
</p><br />
</div><br />
<br />
<br />
<div class="mainbody mainbody6" id="label_6"><br />
<p class="atop" id="cite"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1] Hanan et al.: Enzyme characterisation and kinetic modelling of the pentose phosphate pathway in yeast, PeerJ PrePrints ,<a href="https://peerj.com/preprints/146v1/"> https://peerj.com/preprints/146v1/</a>, v1 received: 9 Dec 2013, published: 9 Dec 2013, doi: 10.7287/peerj.preprints.146v1<br/><br />
[2]<a href="http://www.ebi.ac.uk/biomodels-main/">http://www.ebi.ac.uk/biomodels-main/</a>, BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models<br/><br />
[3]Smallbone et al.: A model of yeast glyolysis based on a consistent kinetic characterisation of all its enzymes, FEBS Letters 587 (2013) 2832–2841<br/><br />
[4] Teusink, B. et al.: Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry, Eur. J. Biochem.267, 5313–5329<br/><br />
[5] Xin-Guang Zhu et al.: A simple model of the Calvin cycle has only one physiologic feasible steady state under the same external conditions, Nonlinear Analysis: Real World Applications 10 (2009) 1490–1499<br/><br />
[6] <a href="http://jjj.mib.ac.uk/database/smallbone">http://jjj.mib.ac.uk/database/smallbone</a>, JWS online-smallbone model<br/><br />
[7] John E Dueber et al.: Synthetic protein scaffolds provide modular control over metabolic flux, NATURE BIOTECHNOLOGY, VOLUME 27 ,NUMBER 8, 753-759<br/><br />
[8] Guadalupe-Medina et al.: Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast, Biotechnology for Biofuels 2013 6:125<br/><br />
</p><br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/OverviewTeam:SCUT/Model/Overview2014-10-18T02:39:58Z<p>Soipan: </p>
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<p onclick="scroll_1()">Background</p><br />
<p onclick="scroll_2()">Carbon dioxide fixed part</p><br />
<p onclick="scroll_3()">n-butanol part</p><br />
</div><br />
<div class="navihead navihead2"><a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
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<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Complete Network</p><br />
<p>The function of Rubisco</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3"><br />
<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
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<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
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<p class="atop"><br />
<span>Background</span><br />
</p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Model-outline.PNG" id="over_right"><br />
The design and redesign is one of the hallmarks of synthesis biology. In order to test the consistence of the pathway we designed and the function of scaffold we used, modeling is the most powerful tool to be used before doing experiments.<br/><br />
</p><br />
</div><br />
<div class="mainbody" id="label_2"><br />
<p class="atop"><br />
<span>Carbon dioxide fixed part</span><br />
</p><br />
<p><br />
For the carbon dioxide fixed part, we use<span> ODEs (ordinary differential equations)</span> to <span>simulate the pathway</span> and proof <span>the function of RuBisCo</span>. With the help of <span>parameter sweep</span>, we find out the <span>optimal reaction rate ratio</span> of the reactions involved in the scaffold. By the way ,we also use <span>the“bottom-up” strategy</span>, the most famous principle of Computer Science, to guide our work. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_3"><br />
<p class="atop"><br />
<span>n-butanol part</span><br />
</p><br />
<p><br />
For the n-butanol part, in order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we construct a model by using <span>Michealis-Menton kinetics</span> and <span>ODEs (ordinary differential equations)</span>. The model shows that, with high concentrations of NADH and NADPH in mitochondria, the production of n-butanol will be greatly improved.<br />
</p><br />
<p><br />
<span>Besides, all of our programs run on the MATLAB.</span><br />
</p><br />
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<p>Achievements>>></p><br />
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<a href="https://2014.igem.org/Team:SCUT/DP/Protocol"><img src="https://static.igem.org/mediawiki/2014/a/af/Datapage3-01.png"></a></a><br />
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<p>Protocol</p><br />
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<span>BRONZE</span><br />
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<p><br />
1.Register the team, SCUT-igemers have spent a great summer, we share our joy and pain, give away our energy and sway our sweat, what we get is an unforgettable experience and precious friendship.<br/><br />
2.Successfully complete and submit this iGEM 2014 Judging form.<br/><br />
3.We have made our own wiki about our project, which records everything we have done this summer, and our wiki is much more attractive than previous igem team.<br/><br />
4.We have made our posters and are going to present a presentation on our project at the iGEM Jamboree in November.<br/><br />
5.We have detailed the division of experiment clearly on our wiki, and most of work is done by students under the guidance of teamleaders and<br />
instructos. Besides, this year we have more sponsors than last year.<br/><br />
6.More than 10 parts have been submitted by the official: <a href="http://parts.igem.org/Part:BBa_K1462090">BBa_K1462090</a>, &nbsp;<a href="http://parts.igem.org/Part:BBa_K1462110">BBa_K1462110</a>, &nbsp;<a href="http://parts.igem.org/Part:BBa_K1462120">BBa_K1462120</a>, &nbsp;<a href="http://parts.igem.org/Part:BBa_K1462130">BBa_K1462130</a>, et al, and the parts we submit perform function properly.<br />
</p><br />
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</div><br />
<br />
<div class="mainbody"><br />
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<span>SILVER</span><br />
</p><br />
<p><br />
1.All the parts we submit can perform function properly<br/><br />
2.We have charaterized the features of our parts in the “Main Page” section of that Part’s/Device’s Registry entry.<br/><br />
3.Submit the new part to the iGEM Parts Registry<br/><br />
4.We have created the safety form on our wiki and our project may have implications for the environment, security, safety and ethics and/or ownership and sharing. <br/><br />
</p><br />
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<p class="atop"><br />
<span>GOLD</span><br />
</p><br />
<p><br />
1.This year,we have worked hard to IMPROVE characterization and ADDED a lot useful information for existing Biobrick. We have tested TEF2<br />
(<a href="http://parts.igem.org/Part:BBa_K1462430">BBa_K1462430</a>)and TDH3(<a href="http://parts.igem.org/Part:BBa_K1462440">BBa_K1462440</a>), quantified Galactose Dose titer to test the induction intensity of GAL1 promter(<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>,<a href="http://parts.igem.org/Part:BBa_K1462450"><br />
BBa_K1462450</a><br />
) and completed decription of a lot existing biobrick (<a href="http://parts.igem.org/Part:BBa_K900003"><br />
BBa_K900003</a>,<a hf="http://parts.igem.org/Part:BBa_K900002"><br />
BBa_K900002</a>,<a href="http://parts.igem.org/Part:BBa_K900005"><br />
BBa_K900005</a>,<a href="http://parts.igem.org/Part:BBa_K1462430"><br />
BBa_K1462430</a>,<br />
<a href="http://parts.igem.org/Part:BBa_K1462440"><br />
BBa_K1462440</a>,<a href="http://parts.igem.org/Part:BBa_J63006"><br />
BBa_J63006</a>)<br/><br />
2.This year, the SCUT 2014 iGEM team have made great efforts to let common people know about Synthetic Biology, including opening serveral lectures for high school students, undergraduates, and professors, traveling to Hongkong, Wuhan, Shenzhen and Guangzhou to communicate and discuss synthetic biology with many other iGEM teams and conducting a questionnaire survey to gather the information of public awareness and approving degree of Synthetic BiologyAdditionally, we issued our original magazines to introduce the knowledge of Synthetic Biology and iGEM, the aim of our project, the prospect of our iGEM life etc.. <br />
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<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/DP/Achievements"><img src="https://static.igem.org/mediawiki/2014/1/1d/Datapage2-01.png"></a><br />
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<p>CO<sub>2</sub> Fixation</p><br />
<p>n-Butanol Prod</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/DP/Protocol"><img src="https://static.igem.org/mediawiki/2014/a/af/Datapage3-01.png"></a><br />
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<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<span>Overview</span><br />
</p><br />
<br />
<p>This summer, we have characterized <b>105</b> BioBricks which could either be used directly or serve as a universal tool readily for potential scientific or engineering use.<br />
</p><br />
<p>Those Biobricks could be divided into 4 groups.<br />
</p><br />
<br />
<span class="xiaobiaoti">n-Butanol Production<span><br />
<br />
<p><a href="http://parts.igem.org/Part:BBa_K1462060"><b>BBa_K1462060</b></a>: adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum. It participates in generation of useful aldehyde, ketone, or alcohol groups during biosynthesis of various metabolites. And sometimes alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+.<br />
</p><br />
<p><br />
<b>BBa_K1462000</b> to <b>BBa_K1462080</b> are basic parts which are basic components of the pathway, including some enzymes and terminators.</br> <br />
<b>BBa_K1462110</b> to <b>BBa_K1462200</b> are proteins fused with GFP used to verify the location of enzymes, in cytoplasm or in mitochondrial matrix.</br> <br />
<b>BBa_K1462210</b> to <b>BBa_K1462210</b> are responsible for the construction of n-butanol biosynthesis pathway in cytoplasm or in mitochondrial matrix. <br />
</p><br />
<br />
CO<sub>2</sub> Fixation<br />
<br />
<p><br />
<a href="http://parts.igem.org/Part:BBa_K1462380"><b>BBa_K1462380</b></a>: CA (carbonic anhydrases or carbonate dehydratases), form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The reaction catalyzed by carbonic anhydrase is: H<sub>2</sub>CO<sub>3</sub>↔CO<sub>2</sub>+ H<sub>2</sub>O<br />
</p><br />
<p><b>BBa_K1462310</b> to <b>BBa_K1462450</b> are BioBricks of CO<sub>2</sub> fixation pathway, which utilize the high-concentration ATP and CO<sub>2</sub> around mitochondria to efficiently fix CO<sub>2</sub> and improve the C sequestration of yeast. This pathway increases the yield of pyruvate for butanol production as the standing point. <a href="http://parts.igem.org/Part:BBa_K1462980"><b>BBa_K1462980</b></a> and <a href="http://parts.igem.org/Part:BBa_K1462990"><b>BBa_K1462990</b></a> are EroGL/EroGS, two E.coli protein-folding chaperons, which stimulate functional expression of RuBisCo.<br />
</p><br />
<br />
Scaffold Proteins<br />
<br />
<p><a href="http://parts.igem.org/Part:BBa_K1462460"><b>BBa_K1462460</b></a>, <a href="http://parts.igem.org/Part:BBa_K1462470"><b>BBa_K1462470</b></a> and <a href="http://parts.igem.org/Part:BBa_K1462480"><b>BBa_K1462480</b></a> are corresponding to the design of GBD, SH3, PDZ ligands; <a href="http://parts.igem.org/Part:BBa_K1462501"><b>BBa_K1462501</b></a>, while <a href="http://parts.igem.org/Part:BBa_K1462502"><b>BBa_K1462502</b></a>, <a href="http://parts.igem.org/Part:BBa_K1462503"><b>BBa_K1462503</b></a> are relevant to the construction of GBD, SH3, PDZ domains with their linkers. <b>BBa_K1462690</b> to <b>BBa_K1462840</b> are different proportions of scaffold proteins, GBD(x), SH3(y) and PDZ(z). Our team concentrates on constructing a tool with a wide range of applications which would beneficial to the researchers from all over the world. With the utilization of the tools, you could control the direction of metabolic flux and enhance the efficiency of biochemical reactions via regulating the ratios of the enzymes and restricting the reactions in a fixed domain. Meanwhile, it achieves the quantification of the projects by altering the number of each enzyme and obtaining the optimum ratio.<br />
</p><br />
<br />
Leading Peptides<br />
<br />
<p><b>BBa_K1462850</b> to <b>BBa_K1462970</b> are leading peptides in yeast. We chose 6 locations in yeast, Plasma Membrane, Vacuolar membrane, Nucleus, Peroxisome, Cytoderm and Endoplasmic reticulum. The verifications of these leading peptides are the first step for us to take further studies in subcellular compartments.<br />
</p><br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_FixationTeam:SCUT/Project/System Construction/Co2 Fixation2014-10-18T00:47:32Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_Fixation"><img src="https://static.igem.org/mediawiki/2014/4/44/Project2-01.png"></a><br />
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<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Design</p><br />
<p onclick="scroll_3()">Construction</p><br />
<p onclick="scroll_4()">Leading Peptide Test</p><br />
<p onclick="scroll_5()">References</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/n-Butanol Prod" id="special" title="A link"><p>n-Butanol Prod>>></p></a><br />
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<div class="navibody navibody2_2" id="navibody2_2_2"><br />
<p>Introduction</p><br />
<p>Design</p><br />
<p>Construction</p><br />
<p>Localization Test</p><br />
<p>References</p><br />
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<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Project/Analysis_and_Discussion"><img src="https://static.igem.org/mediawiki/2014/f/fe/Project3-01.png"></a><br />
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<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<span>Introduction</span><br />
</p><br />
<p>The components of the enzyme machinery anchored onto the outer mitochondrial membrane were selected within the known endogenous metabolic enzymes of S. cerevisiae and other exogenous enzymes, considering the high availability of ATP and CO<sub>2</sub> around mitochodria. The selected enzymes were PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) that working sequentially improve the carbon sequestration of the yeast,or in other words, increase the yield of pyruvate thereby increasing butanol production. To maximize the use of ATP and CO<sub>2</sub>, a selected leading peptide (Tom22) was used to anchor three exogenous scaffold proteins (GBD, SH3 and PDZ) able to bind the above-mentioned enzymes within a limited distance from the membrane.<br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/parts/0/08/Rubisco-cut.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 1 | The overview of our project.</b> </p><br />
<br />
</p><br />
<br />
<span class="xiaobiaoti">PRK</span><br />
<br />
<p><br />
A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:<br />
<br/><br />
<b>ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate</b><br />
<br/><br/><br />
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.<br />
<br/><br/><br />
The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway. <br />
<br/><br/><br />
</p><br />
<span class="xiaobiaoti">RuBisCo</span><br />
<p><br />
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. <br />
<br/><br />
<b>Ribulose 1,5-bisphosphate + CO<sub>2</sub> → 2x 3-phosphoglycerate</b><br />
<br/><br/><br />
In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O<sub>2</sub>) instead of carbon dioxide (CO<sub>2</sub>). Thus, something must be taken to increase concentration of the substrate of interest (CO<sub>2</sub>)to bring a high yield. <br />
<br/><br/><br />
The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway<br />
</p><br />
<span class="xiaobiaoti">CA</span><br />
<br />
<p>Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide<br />
and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes.<br />
The reaction catalyzed by carbonic anhydrase is:<br />
<br/><br />
<b>H<sub>2</sub>CO<sub>3</sub> → CO<sub>2</sub> + H<sub>2</sub>O</b><br />
<br/><br/><br />
<br />
The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.<br />
<br/><br/><br />
The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.<br />
</p><br />
<br />
<span class="xiaobiaoti">Chaperons</span><br />
<p>It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol. <br />
<br/><br/><img src="https://static.igem.org/mediawiki/parts/1/1e/Molecular_chaperone.jpg"><br />
<p style="margin-top:10px;"><b><br />
Figure 2 | The schematic of mechanism of the molecular chaperons GroES and GroEL.</b> Firstly, an ATP binds with GroEL and the ATP-GroEL immediately captures the other unit GroES and there comes the GroEL-GroES-ATP complex.With the help of the complex, the unfolded RuBiSco protein can then properly fold and make a difference in our project.<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Scaffold Protein</span><br />
<p><br />
Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.<br />
<br/><br/><br />
Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/1a/TOM22.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 3 | </b>As shown in the picture, we construct the scaffold protein fusing with the Tom22 which is the member of the Tom family. Tom family is the protein complex that helps the protein including leading peptide enter into the mitochondria. We utilized the embed protein Tom22 to make our scaffold protein (the scaffold protein fuse with the Tom22).<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Leading Peptides</span><br />
<p><br />
In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.<br />
</p><br />
<br />
<br />
</div><br />
<div class="mainbody" id="label_2"><br />
<p class="atop"><br />
<span>Design</span><br />
</p><br />
<p><br />
Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.<br />
</p><br />
<p><br />
We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.<br />
</p><br />
<p><br />
The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.<br />
</p><br />
<p><br />
It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in <a href="https://2014.igem.org/Team:SCUT/Project/Other_Work">Quantification of Promoter Activities</a>).<br />
</p><br />
<br />
</div><br />
<div class="mainbody" id="label_3"><br />
<p class="atop"><br />
<span>Construction</span><br />
</p><br />
<p>The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.<br />
We combine not only our enzymes PRK, RuBiSco and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBiSco and CA</p><p><br />
These are our construction of our pathway and scaffold protein.<br />
<img src="https://static.igem.org/mediawiki/parts/d/d8/Plasmid-firedfish.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 4 |</b> We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA are built up in the plasmid Yeplac181. Moreover, the FP + ligand is also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid Yep352<br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/19/Bigna.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Fiugre 5 | </b>The SCUT A is the pathway that the RuBiSco contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.<br />
</p><img src="https://static.igem.org/mediawiki/parts/0/0b/Fermentation.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 6 | At the pre-experiment</b>, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other(group Ⅱ) consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis.<br />
As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells(group 0). With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_4"><br />
<p class="atop"><br />
<span>Leading Peptide Testing</span><br />
</p><br />
<p>We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.<br />
</p><p><br />
<img src="https://static.igem.org/mediawiki/parts/8/85/Bgy.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 7 | </b>This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial <br />
membrane.<br />
</p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/2BGY.jpg"></p><br />
<p style="margin-top:10px;"><br />
<b>Figure 8 | </b> The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.<br />
</p><br />
<br />
</p><br />
</p><br />
</div><br />
<div class="mainbody" id="label_5"><br />
<p class="atop"><br />
<span>References</span><br />
</p><br />
<p>[1] Hillel K. Brandes‡, Fred C. Hartman§¶, Tse-Yuan S. Lu§, et al. : <b>Efficient Expression of the Gene for Spinach Phosphoribulokinase in Pichia pastoris and Utilization of the Recombinant Enzyme to Explore the Role of Regulatory Cysteinyl Residues by Site-directed Mutagenesis*.</b> The Journal of Biological Chemistry 1996 Vol. 271, No. 11, Issue of March 15, pp. 6490–6496.<br />
</p><p><br />
[2] F. Robert Tabita, Sriram Satagopan, Thomas E. Hanson, et al. : <b>Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.</b> Journal of Experimental Botany 2008 Vol. 59, No. 7, pp. 1515–1524.<br />
</p><p><br />
[3] Christopher R. Somerville and Shauna C. Somerville: <b>Cloning and Expression of the Rhodospirillum rubrum Ribulosebisphosphate Carboxylase Gene in E. coil.</b> Mol Gen Genet 1984 193:214-219<br />
</p><p><br />
[4] Víctor Guadalupe-Medina, H Wouter Wisselink, Marijke AH Luttik, et al. : <b>Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast.</b> Biotechnology for Biofuels 2013 6:125.<br />
</p><p><br />
[5] Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, et al. : <b>Modularity of a carbon-fixing protein organelle.</b> PNAS 2012 vol. 109 , no. 2: 478-483.<br />
</p><p><br />
[6] Jian Qiu, Lena-Sophie Wenz, Ralf M. Zerbes, et al. : <b>Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation.</b> Cell 2013 154, 596–608.<br />
</p><p><br />
[7] Birgitta M. GEIER', IIermann SCHAGGER', Claus ORTWEIN", et al. : <b>Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated.</b> Eur. J. Biochem. 227, 296-302 (1995). Eur. J. Biochem 1995 227, 296-302.<br />
</p><p><br />
[8] MA Jun, SUN Fei: <b>Translocation of Mitochondrial Proteins.</b> ACTA BIOPHYSICA SINICA 2010 Vol.26 No.10: 880-893<br />
</p><p><br />
[9] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al. : <b>Synthetic protein scaffolds provide modular control.</b> NATURE BIOTECHNOLOGY 2009 Vol. 27 No. 8<br />
</p><p><br />
[10] Matthew C. Good, Jesse G. Zalatan, Wendell A. Lim† : <b>Scaffold Proteins: Hubs for Controlling the Flow of Cellular.</b> Science 2011 332, 680.<br />
</p><p><br />
[11] Tae SeokMoona, JohnE.Dueber, EricShiue, et al. : <b>Use of modular, synthetic scaffolds forim proved production of glucaricacid in engineered E. coli over metabolic flux.</b> Metabolic Engineering 2010 12 298–305. </p><br />
<br />
</div><br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_FixationTeam:SCUT/Project/System Construction/Co2 Fixation2014-10-18T00:44:28Z<p>Soipan: </p>
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<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Design</p><br />
<p onclick="scroll_3()">Construction</p><br />
<p onclick="scroll_4()">Leading Peptide Test</p><br />
<p onclick="scroll_5()">References</p><br />
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</div><br />
<div class="navibody navibody2_2" id="navibody2_2_2"><br />
<p>Introduction</p><br />
<p>Design</p><br />
<p>Construction</p><br />
<p>Localization Test</p><br />
<p>References</p><br />
</div><br />
</div><br />
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</div><br />
<div class="navibody navibody3"><br />
<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
</div><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<br />
<!--可编辑区域--><br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p>The components of the enzyme machinery anchored onto the outer mitochondrial membrane were selected within the known endogenous metabolic enzymes of S. cerevisiae and other exogenous enzymes, considering the high availability of ATP and CO<sub>2</sub> around mitochodria. The selected enzymes were PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) that working sequentially improve the carbon sequestration of the yeast,or in other words, increase the yield of pyruvate thereby increasing butanol production. To maximize the use of ATP and CO<sub>2</sub>, a selected leading peptide (Tom22) was used to anchor three exogenous scaffold proteins (GBD, SH3 and PDZ) able to bind the above-mentioned enzymes within a limited distance from the membrane.<br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/parts/0/08/Rubisco-cut.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 1 | The overview of our project.</b> </p><br />
<br />
</p><br />
<br />
<span class="xiaobiaoti">PRK</span><br />
<br />
<p><br />
A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:<br />
<br/><br />
<b>ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate</b><br />
<br/><br/><br />
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.<br />
<br/><br/><br />
The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway. <br />
<br/><br/><br />
</p><br />
<span class="xiaobiaoti">RuBisCo</span><br />
<p><br />
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. <br />
<br/><br />
<b>Ribulose 1,5-bisphosphate + CO<sub>2</sub> → 2x 3-phosphoglycerate</b><br />
<br/><br/><br />
In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O<sub>2</sub>) instead of carbon dioxide (CO<sub>2</sub>). Thus, something must be taken to increase concentration of the substrate of interest (CO<sub>2</sub>)to bring a high yield. <br />
<br/><br/><br />
The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway<br />
</p><br />
<span class="xiaobiaoti">CA</span><br />
<br />
<p>Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide<br />
and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes.<br />
The reaction catalyzed by carbonic anhydrase is:<br />
<br/><br />
<b>H<sub>2</sub>CO<sub>3</sub> → CO<sub>2</sub> + H<sub>2</sub>O</b><br />
<br/><br/><br />
<br />
The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.<br />
<br/><br/><br />
The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.<br />
</p><br />
<br />
<span class="xiaobiaoti">Chaperons</span><br />
<p>It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol. <br />
<br/><br/><img src="https://static.igem.org/mediawiki/parts/1/1e/Molecular_chaperone.jpg"><br />
<p style="margin-top:10px;"><b><br />
Figure 2 | The schematic of mechanism of the molecular chaperons GroES and GroEL.</b> Firstly, an ATP binds with GroEL and the ATP-GroEL immediately captures the other unit GroES and there comes the GroEL-GroES-ATP complex.With the help of the complex, the unfolded RuBiSco protein can then properly fold and make a difference in our project.<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Scaffold Protein</span><br />
<p><br />
Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.<br />
<br/><br/><br />
Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/1a/TOM22.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 3 | </b>As shown in the picture, we construct the scaffold protein fusing with the Tom22 which is the member of the Tom family. Tom family is the protein complex that helps the protein including leading peptide enter into the mitochondria. We utilized the embed protein Tom22 to make our scaffold protein (the scaffold protein fuse with the Tom22).<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Leading Peptides</span><br />
<p><br />
In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.<br />
</p><br />
<br />
<br />
</div><br />
<div class="mainbody" id="label_2"><br />
<p class="atop"><br />
<span>Design</span><br />
</p><br />
<p><br />
Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.<br />
</p><br />
<p><br />
We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.<br />
</p><br />
<p><br />
The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.<br />
</p><br />
<p><br />
It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in <a href="https://2014.igem.org/Team:SCUT/Project/Other_Work">Quantification of Promoter Activities</a>).<br />
</p><br />
<br />
</div><br />
<div class="mainbody" id="label_3"><br />
<p class="atop"><br />
<span>Construction</span><br />
</p><br />
<p>The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.<br />
We combine not only our enzymes PRK, RuBiSco and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBiSco and CA</p><p><br />
These are our construction of our pathway and scaffold protein.<br />
<img src="https://static.igem.org/mediawiki/parts/d/d8/Plasmid-firedfish.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 4 |</b> We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA are built up in the plasmid Yeplac181. Moreover, the FP + ligand is also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid Yep352<br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/19/Bigna.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Fiugre 5 | </b>The SCUT A is the pathway that the RuBiSco contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.<br />
</p><img src="https://static.igem.org/mediawiki/parts/0/0b/Fermentation.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 6 | At the pre-experiment</b>, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other(group Ⅱ) consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis.<br />
As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells(group 0). With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_4"><br />
<p class="atop"><br />
<span>Leading Peptide Testing</span><br />
</p><br />
<p>We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.<br />
</p><p><br />
<img src="https://static.igem.org/mediawiki/parts/8/85/Bgy.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 7 | </b>This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial <br />
membrane.<br />
</p><br />
<p><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/2BGY.jpg"></p><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 8 | </b> The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.<br />
</p><br />
<br />
</p><br />
</p><br />
</div><br />
<div class="mainbody" id="label_5"><br />
<p class="atop"><br />
<span>References</span><br />
</p><br />
<p>[1] Hillel K. Brandes‡, Fred C. Hartman§¶, Tse-Yuan S. Lu§, et al. : <b>Efficient Expression of the Gene for Spinach Phosphoribulokinase in Pichia pastoris and Utilization of the Recombinant Enzyme to Explore the Role of Regulatory Cysteinyl Residues by Site-directed Mutagenesis*.</b> The Journal of Biological Chemistry 1996 Vol. 271, No. 11, Issue of March 15, pp. 6490–6496.<br />
</p><p><br />
[2] F. Robert Tabita, Sriram Satagopan, Thomas E. Hanson, et al. : <b>Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.</b> Journal of Experimental Botany 2008 Vol. 59, No. 7, pp. 1515–1524.<br />
</p><p><br />
[3] Christopher R. Somerville and Shauna C. Somerville: <b>Cloning and Expression of the Rhodospirillum rubrum Ribulosebisphosphate Carboxylase Gene in E. coil.</b> Mol Gen Genet 1984 193:214-219<br />
</p><p><br />
[4] Víctor Guadalupe-Medina, H Wouter Wisselink, Marijke AH Luttik, et al. : <b>Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast.</b> Biotechnology for Biofuels 2013 6:125.<br />
</p><p><br />
[5] Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, et al. : <b>Modularity of a carbon-fixing protein organelle.</b> PNAS 2012 vol. 109 , no. 2: 478-483.<br />
</p><p><br />
[6] Jian Qiu, Lena-Sophie Wenz, Ralf M. Zerbes, et al. : <b>Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation.</b> Cell 2013 154, 596–608.<br />
</p><p><br />
[7] Birgitta M. GEIER', IIermann SCHAGGER', Claus ORTWEIN", et al. : <b>Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated.</b> Eur. J. Biochem. 227, 296-302 (1995). Eur. J. Biochem 1995 227, 296-302.<br />
</p><p><br />
[8] MA Jun, SUN Fei: <b>Translocation of Mitochondrial Proteins.</b> ACTA BIOPHYSICA SINICA 2010 Vol.26 No.10: 880-893<br />
</p><p><br />
[9] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al. : <b>Synthetic protein scaffolds provide modular control.</b> NATURE BIOTECHNOLOGY 2009 Vol. 27 No. 8<br />
</p><p><br />
[10] Matthew C. Good, Jesse G. Zalatan, Wendell A. Lim† : <b>Scaffold Proteins: Hubs for Controlling the Flow of Cellular.</b> Science 2011 332, 680.<br />
</p><p><br />
[11] Tae SeokMoona, JohnE.Dueber, EricShiue, et al. : <b>Use of modular, synthetic scaffolds forim proved production of glucaricacid in engineered E. coli over metabolic flux.</b> Metabolic Engineering 2010 12 298–305. </p><br />
<br />
</div><br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_FixationTeam:SCUT/Project/System Construction/Co2 Fixation2014-10-18T00:41:31Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Design</p><br />
<p onclick="scroll_3()">Construction</p><br />
<p onclick="scroll_4()">Leading Peptide Test</p><br />
<p onclick="scroll_5()">References</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/n-Butanol Prod" id="special" title="A link"><p>n-Butanol Prod>>></p></a><br />
</div><br />
<div class="navibody navibody2_2" id="navibody2_2_2"><br />
<p>Introduction</p><br />
<p>Design</p><br />
<p>Construction</p><br />
<p>Localization Test</p><br />
<p>References</p><br />
</div><br />
</div><br />
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</div><br />
<div class="navibody navibody3"><br />
<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<br />
<!--可编辑区域--><br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p>The components of the enzyme machinery anchored onto the outer mitochondrial membrane were selected within the known endogenous metabolic enzymes of S. cerevisiae and other exogenous enzymes, considering the high availability of ATP and CO<sub>2</sub> around mitochodria. The selected enzymes were PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) that working sequentially improve the carbon sequestration of the yeast,or in other words, increase the yield of pyruvate thereby increasing butanol production. To maximize the use of ATP and CO<sub>2</sub>, a selected leading peptide (Tom22) was used to anchor three exogenous scaffold proteins (GBD, SH3 and PDZ) able to bind the above-mentioned enzymes within a limited distance from the membrane.<br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/parts/0/08/Rubisco-cut.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 1 | The overview of our project.</b> </p><br />
<br />
</p><br />
<br />
<span class="xiaobiaoti">PRK</span><br />
<br />
<p><br />
A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:<br />
<br/><br />
<b>ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate</b><br />
<br/><br/><br />
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.<br />
<br/><br/><br />
The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway. <br />
<br/><br/><br />
</p><br />
<span class="xiaobiaoti">RuBisCo</span><br />
<p><br />
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. <br />
<br/><br />
<b>Ribulose 1,5-bisphosphate + CO<sub>2</sub> → 2x 3-phosphoglycerate</b><br />
<br/><br/><br />
In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O<sub>2</sub>) instead of carbon dioxide (CO<sub>2</sub>). Thus, something must be taken to increase concentration of the substrate of interest (CO<sub>2</sub>)to bring a high yield. <br />
<br/><br/><br />
The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway<br />
</p><br />
<span class="xiaobiaoti">CA</span><br />
<br />
<p>Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide<br />
and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes.<br />
The reaction catalyzed by carbonic anhydrase is:<br />
<br/><br />
<b>H<sub>2</sub>CO<sub>3</sub> → CO<sub>2</sub> + H<sub>2</sub>O</b><br />
<br/><br/><br />
<br />
The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.<br />
<br/><br/><br />
The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.<br />
</p><br />
<br />
<span class="xiaobiaoti">Chaperons</span><br />
<p>It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol. <br />
<br/><br/><img src="https://static.igem.org/mediawiki/parts/1/1e/Molecular_chaperone.jpg"><br />
<p style="margin-top:10px;"><b><br />
Figure 2 | The schematic of mechanism of the molecular chaperons GroES and GroEL.</b> Firstly, an ATP binds with GroEL and the ATP-GroEL immediately captures the other unit GroES and there comes the GroEL-GroES-ATP complex.With the help of the complex, the unfolded RuBiSco protein can then properly fold and make a difference in our project.<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Scaffold Protein</span><br />
<p><br />
Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.<br />
<br/><br/><br />
Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/1a/TOM22.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 3 | </b>As shown in the picture, we construct the scaffold protein fusing with the Tom22 which is the member of the Tom family. Tom family is the protein complex that helps the protein including leading peptide enter into the mitochondria. We utilized the embed protein Tom22 to make our scaffold protein (the scaffold protein fuse with the Tom22).<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Leading Peptides</span><br />
<p><br />
In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.<br />
</p><br />
<br />
<br />
</div><br />
<div class="mainbody" id="label_2"><br />
<p class="atop"><br />
<span>Design</span><br />
</p><br />
<p><br />
Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.<br />
</p><br />
<p><br />
We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.<br />
</p><br />
<p><br />
The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.<br />
</p><br />
<p><br />
It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in <a href="https://2014.igem.org/Team:SCUT/Project/Other_Work">Quantification of Promoter Activities</a>).<br />
</p><br />
<br />
</div><br />
<div class="mainbody" id="label_3"><br />
<p class="atop"><br />
<span>Construction</span><br />
</p><br />
<p>The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.<br />
We combine not only our enzymes PRK, RuBiSco and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBiSco and CA</p><p><br />
These are our construction of our pathway and scaffold protein.<br />
<img src="https://static.igem.org/mediawiki/parts/d/d8/Plasmid-firedfish.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 4 |</b> We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA are built up in the plasmid Yeplac181. Moreover, the FP + ligand is also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid Yep352<br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/19/Bigna.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Fiugre 5 | </b>The SCUT A is the pathway that the RuBiSco contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.<br />
</p><img src="https://static.igem.org/mediawiki/parts/0/0b/Fermentation.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 6 | At the pre-experiment</b>, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other(group Ⅱ) consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis.<br />
As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells(group 0). With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_4"><br />
<p class="atop"><br />
<span>Leading Peptide Testing</span><br />
</p><br />
<p>We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.<br />
</p><p><br />
<img src="https://static.igem.org/mediawiki/parts/8/85/Bgy.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 7 | </b>This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial <br />
membrane.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/2BGY.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 8 | </b> The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.<br />
</p><br />
<br />
</p><br />
</p><br />
</div><br />
<div class="mainbody" id="label_5"><br />
<p class="atop"><br />
<span>References</span><br />
</p><br />
<p>[1] Hillel K. Brandes‡, Fred C. Hartman§¶, Tse-Yuan S. Lu§, et al. : <b>Efficient Expression of the Gene for Spinach Phosphoribulokinase in Pichia pastoris and Utilization of the Recombinant Enzyme to Explore the Role of Regulatory Cysteinyl Residues by Site-directed Mutagenesis*.</b> The Journal of Biological Chemistry 1996 Vol. 271, No. 11, Issue of March 15, pp. 6490–6496.<br />
</p><p><br />
[2] F. Robert Tabita, Sriram Satagopan, Thomas E. Hanson, et al. : <b>Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.</b> Journal of Experimental Botany 2008 Vol. 59, No. 7, pp. 1515–1524.<br />
</p><p><br />
[3] Christopher R. Somerville and Shauna C. Somerville: <b>Cloning and Expression of the Rhodospirillum rubrum Ribulosebisphosphate Carboxylase Gene in E. coil.</b> Mol Gen Genet 1984 193:214-219<br />
</p><p><br />
[4] Víctor Guadalupe-Medina, H Wouter Wisselink, Marijke AH Luttik, et al. : <b>Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast.</b> Biotechnology for Biofuels 2013 6:125.<br />
</p><p><br />
[5] Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, et al. : <b>Modularity of a carbon-fixing protein organelle.</b> PNAS 2012 vol. 109 , no. 2: 478-483.<br />
</p><p><br />
[6] Jian Qiu, Lena-Sophie Wenz, Ralf M. Zerbes, et al. : <b>Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation.</b> Cell 2013 154, 596–608.<br />
</p><p><br />
[7] Birgitta M. GEIER', IIermann SCHAGGER', Claus ORTWEIN", et al. : <b>Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated.</b> Eur. J. Biochem. 227, 296-302 (1995). Eur. J. Biochem 1995 227, 296-302.<br />
</p><p><br />
[8] MA Jun, SUN Fei: <b>Translocation of Mitochondrial Proteins.</b> ACTA BIOPHYSICA SINICA 2010 Vol.26 No.10: 880-893<br />
</p><p><br />
[9] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al. : <b>Synthetic protein scaffolds provide modular control.</b> NATURE BIOTECHNOLOGY 2009 Vol. 27 No. 8<br />
</p><p><br />
[10] Matthew C. Good, Jesse G. Zalatan, Wendell A. Lim† : <b>Scaffold Proteins: Hubs for Controlling the Flow of Cellular.</b> Science 2011 332, 680.<br />
</p><p><br />
[11] Tae SeokMoona, JohnE.Dueber, EricShiue, et al. : <b>Use of modular, synthetic scaffolds forim proved production of glucaricacid in engineered E. coli over metabolic flux.</b> Metabolic Engineering 2010 12 298–305. </p><br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Project/System_Construction/Co2_FixationTeam:SCUT/Project/System Construction/Co2 Fixation2014-10-18T00:40:29Z<p>Soipan: </p>
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<p>Overview>>></p><br />
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<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Design</p><br />
<p onclick="scroll_3()">Construction</p><br />
<p onclick="scroll_4()">Leading Peptide Test</p><br />
<p onclick="scroll_5()">References</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/System_Construction/n-Butanol Prod" id="special" title="A link"><p>n-Butanol Prod>>></p></a><br />
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<div class="navibody navibody2_2" id="navibody2_2_2"><br />
<p>Introduction</p><br />
<p>Design</p><br />
<p>Construction</p><br />
<p>Localization Test</p><br />
<p>References</p><br />
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<p>Introduction</p><br />
<p>Simulation</p><br />
<p>Reference</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Project/Other_Work"><img src="https://static.igem.org/mediawiki/2014/4/48/Project4-01.png"></a><br />
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<p>Outline</p><br />
<p>Reference</p><br />
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<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p>The components of the enzyme machinery anchored onto the outer mitochondrial membrane were selected within the known endogenous metabolic enzymes of S. cerevisiae and other exogenous enzymes, considering the high availability of ATP and CO<sub>2</sub> around mitochodria. The selected enzymes were PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) that working sequentially improve the carbon sequestration of the yeast,or in other words, increase the yield of pyruvate thereby increasing butanol production. To maximize the use of ATP and CO<sub>2</sub>, a selected leading peptide (Tom22) was used to anchor three exogenous scaffold proteins (GBD, SH3 and PDZ) able to bind the above-mentioned enzymes within a limited distance from the membrane.<br />
</p><br />
<p id="equ"><img src="https://static.igem.org/mediawiki/parts/0/08/Rubisco-cut.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 1 | The overview of our project.</b> </p><br />
<br />
</p><br />
<br />
<span class="xiaobiaoti">PRK</span><br />
<br />
<p><br />
A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:<br />
<br/><br />
<b>ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate</b><br />
<br/><br/><br />
This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.<br />
<br/><br/><br />
The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway. <br />
<br/><br/><br />
</p><br />
<span class="xiaobiaoti">RuBisCo</span><br />
<p><br />
Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose. <br />
<br/><br />
<b>Ribulose 1,5-bisphosphate + CO<sub>2</sub> → 2x 3-phosphoglycerate</b><br />
<br/><br/><br />
In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O<sub>2</sub>) instead of carbon dioxide (CO<sub>2</sub>). Thus, something must be taken to increase concentration of the substrate of interest (CO<sub>2</sub>)to bring a high yield. <br />
<br/><br/><br />
The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway<br />
</p><br />
<span class="xiaobiaoti">CA</span><br />
<br />
<p>Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide<br />
and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes.<br />
The reaction catalyzed by carbonic anhydrase is:<br />
<br/><br />
<b>H<sub>2</sub>CO<sub>3</sub> → CO<sub>2</sub> + H<sub>2</sub>O</b><br />
<br/><br/><br />
<br />
The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.<br />
<br/><br/><br />
The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.<br />
</p><br />
<br />
<span class="xiaobiaoti">Chaperons</span><br />
<p>It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol. <br />
<br/><br/><img src="https://static.igem.org/mediawiki/parts/1/1e/Molecular_chaperone.jpg"><br />
<p style="margin-top:10px;"><b><br />
Figure 2 | The schematic of mechanism of the molecular chaperons GroES and GroEL.</b> Firstly, an ATP binds with GroEL and the ATP-GroEL immediately captures the other unit GroES and there comes the GroEL-GroES-ATP complex.With the help of the complex, the unfolded RuBiSco protein can then properly fold and make a difference in our project.<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Scaffold Protein</span><br />
<p><br />
Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.<br />
<br/><br/><br />
Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/1a/TOM22.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 3 | </b>As shown in the picture, we construct the scaffold protein fusing with the Tom22 which is the member of the Tom family. Tom family is the protein complex that helps the protein including leading peptide enter into the mitochondria. We utilized the embed protein Tom22 to make our scaffold protein (the scaffold protein fuse with the Tom22).<br />
</p><br />
</p><br />
<br />
<span class="xiaobiaoti">Leading Peptides</span><br />
<p><br />
In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.<br />
</p><br />
<br />
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<div class="mainbody" id="label_2"><br />
<p class="atop"><br />
<span>Design</span><br />
</p><br />
<p><br />
Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.<br />
</p><br />
<p><br />
We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.<br />
</p><br />
<p><br />
The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.<br />
</p><br />
<p><br />
It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in <a href="https://2014.igem.org/Team:SCUT/Project/Other_Work">Quantification of Promoter Activities</a>).<br />
</p><br />
<br />
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<div class="mainbody" id="label_3"><br />
<p class="atop"><br />
<span>Construction</span><br />
</p><br />
<p>The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.<br />
We combine not only our enzymes PRK, RuBiSco and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBiSco and CA</p><p><br />
These are our construction of our pathway and scaffold protein.<br />
<img src="https://static.igem.org/mediawiki/parts/d/d8/Plasmid-firedfish.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 4 |</b> We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA are built up in the plasmid Yeplac181. Moreover, the FP + ligand is also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid Yep352<br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/parts/1/19/Bigna.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Fiugre 5 | </b>The SCUT A is the pathway that the RuBiSco contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.<br />
</p><img src="https://static.igem.org/mediawiki/parts/0/0b/Fermentation.jpg"><br />
<p style="margin-top:10px;"><br />
<b>Figure 6 | At the pre-experiment</b>, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other(group Ⅱ) consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis.<br />
As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells(group 0). With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased. <br />
</p><br />
</div><br />
<div class="mainbody" id="label_4"><br />
<p class="atop"><br />
<span>Leading Peptide Testing</span><br />
</p><br />
<p>We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.<br />
</p><p><br />
<img src="https://static.igem.org/mediawiki/parts/8/85/Bgy.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 7 | </b>This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial <br />
membrane.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/2BGY.jpg"><br/><br />
<p style="margin-top:10px;"><br />
<b>Figure 8 | </b> The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.<br />
</p><br />
<br />
</p><br />
</p><br />
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<div class="mainbody" id="label_5"><br />
<p class="atop"><br />
<span>References</span><br />
</p><br />
<p>[1] Hillel K. Brandes‡, Fred C. Hartman§¶, Tse-Yuan S. Lu§, et al. : <b>Efficient Expression of the Gene for Spinach Phosphoribulokinase in Pichia pastoris and Utilization of the Recombinant Enzyme to Explore the Role of Regulatory Cysteinyl Residues by Site-directed Mutagenesis*.</b> The Journal of Biological Chemistry 1996 Vol. 271, No. 11, Issue of March 15, pp. 6490–6496.<br />
</p><p><br />
[2] F. Robert Tabita, Sriram Satagopan, Thomas E. Hanson, et al. : <b>Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.</b> Journal of Experimental Botany 2008 Vol. 59, No. 7, pp. 1515–1524.<br />
</p><p><br />
[3] Christopher R. Somerville and Shauna C. Somerville: <b>Cloning and Expression of the Rhodospirillum rubrum Ribulosebisphosphate Carboxylase Gene in E. coil.</b> Mol Gen Genet 1984 193:214-219<br />
</p><p><br />
[4] Víctor Guadalupe-Medina, H Wouter Wisselink, Marijke AH Luttik, et al. : <b>Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast.</b> Biotechnology for Biofuels 2013 6:125.<br />
</p><p><br />
[5] Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, et al. : <b>Modularity of a carbon-fixing protein organelle.</b> PNAS 2012 vol. 109 , no. 2: 478-483.<br />
</p><p><br />
[6] Jian Qiu, Lena-Sophie Wenz, Ralf M. Zerbes, et al. : <b>Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation.</b> Cell 2013 154, 596–608.<br />
</p><p><br />
[7] Birgitta M. GEIER', IIermann SCHAGGER', Claus ORTWEIN", et al. : <b>Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated.</b> Eur. J. Biochem. 227, 296-302 (1995). Eur. J. Biochem 1995 227, 296-302.<br />
</p><p><br />
[8] MA Jun, SUN Fei: <b>Translocation of Mitochondrial Proteins.</b> ACTA BIOPHYSICA SINICA 2010 Vol.26 No.10: 880-893<br />
</p><p><br />
[9] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al. : <b>Synthetic protein scaffolds provide modular control.</b> NATURE BIOTECHNOLOGY 2009 Vol. 27 No. 8<br />
</p><p><br />
[10] Matthew C. Good, Jesse G. Zalatan, Wendell A. Lim† : <b>Scaffold Proteins: Hubs for Controlling the Flow of Cellular.</b> Science 2011 332, 680.<br />
</p><p><br />
[11] Tae SeokMoona, JohnE.Dueber, EricShiue, et al. : <b>Use of modular, synthetic scaffolds forim proved production of glucaricacid in engineered E. coli over metabolic flux.</b> Metabolic Engineering 2010 12 298–305. </p><br />
<br />
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</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Team/AttributionsTeam:SCUT/Team/Attributions2014-10-18T00:36:41Z<p>Soipan: </p>
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<p>Introdution</p><br />
<p>CO<sub>2</sub> fixed simulation lab </p><br />
<p>Butanol lab notes</p><br />
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<a href="https://2014.igem.org/Team:SCUT/Team/Acknowledgement"><img src="https://static.igem.org/mediawiki/2014/6/6f/Team4-01.png"></a><br />
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<div class="navibody navibody4"><br />
<p>Acknowledgements>>></p><br />
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<a href="https://2014.igem.org/Team:SCUT/Team/Our_University"><img src="https://static.igem.org/mediawiki/2014/3/3b/Team5-01.png"></a><br />
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<div class="navibody navibody5"><br />
<p>SCUT</p><br />
<p>Our University</p><br />
</div><br />
</div><br />
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<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Attributions</span><br />
</p><br />
<p class="first"><br />
<span class="xiaobiaoti">Zhang Junjie </span><br/><br/><br />
The leader of SCUT iGEM team, was responsible for constructing and managing the team, instructing team members to design and implement the project. He was also in charge of contacting with professors and seniors for us, and supervised the progress of all parts and tasks. <br />
</p><br />
<p><br />
<span class="xiaobiaoti">Butanol Part</span><br/><br/><br />
Fan Chuyao, Hu Weipeng were involved in butanol part and made great contributions to constructing and optimizing the butanol pathway in inter-mitochondrial and cytoplasm.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">CO<sub>2</sub> Fixation Part</span><br/><br/><br />
Peng Ye, Chen Zhanru, Yang Bina managed CO<sub>2</sub> fixation part. Peng Ye was in charge of PRK, Rubisco and CA enzyme, Wang Zhizheng also made efforts to assist Peng Ye. Chen ZhanRu made brilliant contributions and devoted himself to testing the scaffold protein using fluorescent protein fused with ligand and he also helped Yang to construct the pathway of scaffold protein. Meanwhile he assisted design group in drawing some vivid pictures. As for Yang Bina, she was diligent and she made great efforts to construct different scaffold protein combinations in order to find the best one. She has created 32 biobricks in total.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Leading Peptide Part</span><br/><br/><br />
Li Linzhou, Zhao Yaran made efforts on leading peptide of subcellular. From searching to experiment planning, they have done all of necessary things. And they really cooperated very well during the whole summer vacation.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Wiki and Logo Design Group</span><br/><br/><br />
Wang Xiuyuan, Ling Zhangyan, and Pan Xueman have undertaken all the tasks of designing of the whole team. They did a lot work including logo, ppt templates, wiki and uniforms. Taking step by step, as a result of clarified division and efficient cooperation , they sophisticatedly design uniforms and posters for the IGEM.Wang feel honored to become one of the IGEMers, in this team, like he said :"I can not only show my talents for editing skills as well as contribute my ideas on design, but also meet with gifted and competent peers here. I feel good to strive for the same goal together."<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Modelling Group</span><br/><br/><br />
Huang Xiaobin, Wu Yuhang and Zhou Shiyan devoted their passion to modeling metabolic pathway to instruct our experiments. This group has a diversified background since the three members come from three different schools. Xiaobin majors in computer science, Yuhang majors in Biotech while Shiyan studies mathematics. Thus they provided more professional works for SCUT iGEM team.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Human Practice</span><br/><br/><br />
The Human Practice part of this year is completely in the charge of Wang Zhizheng. He has determined the topic of HP, “Synthetic Biology Beautifies Our Life”. He designed a variety of methods to promote this topic and make knowledge of synthetic biology and iGEM. He has opened the internet communication platform, compiled our original magazine,”iGEM Life”, traveled to HongKong, Shenzhen, Wuhan and some places to communicate with other iGEM team. What’s more, he and his assistants also did two surveys to observe the change of others' attitude towards synthetic biology and iGEM. So he made considerable efforts and contributions to making HP part better.<br />
</p><br />
</div><br />
<br />
<br />
</body><br />
<br />
<br />
<br />
<br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:22:24Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
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<div id="nb"><br />
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<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:28px;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:21:58Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:30px;width:100%;height:auto;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:21:41Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:30px;width:101%;height:auto;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:21:28Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:30px;width:105%;height:auto;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:21:13Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:30px;width:120%;height:auto;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:20:06Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
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<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png" style="margin-left:30px;"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling006.pngFile:Modeling006.png2014-10-18T00:17:26Z<p>Soipan: uploaded a new version of &quot;File:Modeling006.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:15:00Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4b/RB.jpg"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:RB.jpgFile:RB.jpg2014-10-18T00:14:03Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/File:Modeling006.pngFile:Modeling006.png2014-10-18T00:10:48Z<p>Soipan: uploaded a new version of &quot;File:Modeling006.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/File:Modeling006.pngFile:Modeling006.png2014-10-18T00:04:58Z<p>Soipan: uploaded a new version of &quot;File:Modeling006.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/File:Modeling002.pngFile:Modeling002.png2014-10-18T00:01:34Z<p>Soipan: uploaded a new version of &quot;File:Modeling002.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-18T00:00:07Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/Modeling004.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/Modeling005.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/Modeling006.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling006.pngFile:Modeling006.png2014-10-17T23:59:01Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:58:43Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/Modeling004.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/Modeling005.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling005.pngFile:Modeling005.png2014-10-17T23:57:58Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:57:37Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/Modeling004.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling004.pngFile:Modeling004.png2014-10-17T23:56:37Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:56:03Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
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<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
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<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/6/6a/Modeling003.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling003.pngFile:Modeling003.png2014-10-17T23:54:40Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/File:Modeling002.pngFile:Modeling002.png2014-10-17T23:47:17Z<p>Soipan: uploaded a new version of &quot;File:Modeling002.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/File:Modeling002.pngFile:Modeling002.png2014-10-17T23:45:35Z<p>Soipan: uploaded a new version of &quot;File:Modeling002.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:45:05Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e2/Modeling000.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2821%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Equ_1_%2822%29.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling000.pngFile:Modeling000.png2014-10-17T23:44:20Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:41:23Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2821%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Equ_1_%2822%29.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling001.pngFile:Modeling001.png2014-10-17T23:40:27Z<p>Soipan: uploaded a new version of &quot;File:Modeling001.png&quot;</p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:36:53Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/Equ_1_%2813%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Modeling002.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2821%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Equ_1_%2822%29.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling002.pngFile:Modeling002.png2014-10-17T23:35:12Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:33:46Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#nb{position:absolute;width:100%;top:300px;left:0px;height:auto;}<br />
#show p:hover{background-color:#ff8a5c;}<br />
</style><br />
</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/Equ_1_%2813%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2821%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Equ_1_%2822%29.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Model/N-butanol_simulationTeam:SCUT/Model/N-butanol simulation2014-10-17T23:31:18Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
{{Template:Team:SCUT/Model/mainhead}}<br />
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</head><br />
<body><br />
<div id="nb"><br />
<br />
<div id="combine"><br />
<div id="left"><br />
<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Overview"><img src="https://static.igem.org/mediawiki/2014/d/dc/1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Background</p><br />
<p>Carbon dioxide fixed part</p><br />
<p>n-butanol simulation</p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Rubisco_simulation"><img src="https://static.igem.org/mediawiki/2014/c/c6/Model2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introduction</p><br />
<p>Individual part</p><br />
<p>Combine part</p><br />
<p>The function of RuBisCo</p><br />
<p>Scaffold</p><br />
<p>Reference</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/N-butanol_simulation"><img src="https://static.igem.org/mediawiki/2014/d/d2/Model3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Introduction</p><br />
<p onclick="scroll_2()">Simulation</p><br />
<p onclick="scroll_3()">Reference</p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Model/Tips_for_other_teams"><img src="https://static.igem.org/mediawiki/2014/a/af/Model4-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>Outline</p><br />
<p>Reference</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Overview</span><br />
</p><br />
<p class="first"><br />
In order to simulate the n-butanol biosynthetic pathway in Saccharomyces cerevisiae mitochondria, we constructed a model by Michealis-Menton kinetics and ordinary differential equation(ODE). The model shows that, with high concentrations of NADH in mitochondria, the production of n-butanol will be greatly improved. <br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody2" id="label_2"><br />
<p class="atop"><br />
<span>Introduction</span><br />
</p><br />
<p class="first"><br />
We firstly constructed the biochemical reactions of n-butanol producing pathway by Michealis-Menton kinetics and then modeled them by kinetics at the beginning of the reactions when no products have been accumulated. Finally, considering the special environment of mitochondria, we did the work about the effects of the concentrations of NADH and NADPH, which is abundant in mitochondria.<br />
</p><br />
</div><br />
<br />
<div class="mainbody mainbody3" id="label_3"><br />
<p class="atop"><br />
<span>Simulation</span><br />
</p><br />
<p class="first"><br />
For thiolase [erg10], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/Equ_1_%2813%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/86/Equ_1_%2814%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2815%29.png"><br />
</p><br />
<p><br />
For 3-hydroxybutyryl-coa dehydrogenase[Hbd], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/9/9c/Equ_1_%2816%29.png"><br/><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Modeling001.png"><br />
</p><br />
<p><br />
For crotonase[crt], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/Equ_1_%2820%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/Equ_1_%2821%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/8/83/Equ_1_%2822%29.png"><br />
</p><br />
<p><br />
For BtCoA dehydrogenase [ccr], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/Equ_1_%2823%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f9/Equ_1_%2824%29.png"><br/><br />
<img src="https://static.igem.org/mediawiki/2014/a/ab/Equ_1_%2825%29.png"><br />
</p><br />
<p><br />
For Bldh, butyraldehyde dehydrogenase [AdH2], the reaction is<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6d/Equ_1_%2826%29.png"><br />
</p><br />
<p><br />
The rate expression is defined as<br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/Equ_1_%2827%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/Equ_1_%2831%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ce/Equ_1_%2828%29.png"><br/><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Equ_1_%2829%29.png"><br />
</p><br />
<p><br />
We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. <br/>For simplify ,we set the concentration of NADH and NADPH to 200μM and 100μM respectively and also consider it as a constant .Concentrations of all other metabolite are set to 0 in the beginning.<br />
</p><br />
</div><br />
<div class="mainbody mainbody4" id="label_4"><br />
<p class="atop"><br />
<span>Result</span><br />
</p><br />
<p class="first" id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/Nb-1.jpg"><br/><br />
Figure 1.Production of n-butanol at different of concentration of enzyme<br />
</p><br />
<p><br />
We set the concentration of AcCoA as a constant to 1000μM, and the concentration of NADH, NAD<sup>+</sup>, NADPH and NADP<sup>+</sup> to 200μM, 200μM and 100μM,100μM respectively. The concentration of other substrate are set to 0 at the beginning of the process. In figure 1, we set the concentration of enzyme range from 0 to 1mM. From the result, we can learn that overexpress the enzyme can increase the production of butanol. <br />
</p><br />
<p id="equ"><br />
<img src="https://static.igem.org/mediawiki/2014/1/10/Nb-2.jpg"><br/><br />
figure 2. Production of n-butanol at different of concentration of NADH and NADPH<br />
</p><br />
<p><br />
We construct the pathway into mitochondria for its high concentration of NADH and NADPH, so in figure 2 we range the concentration of NADH and NADPH from 50-200μM, 20-100μM respectively. The result shows that as the concentration is increased, the production can be improved.<br />
</p><br />
</div><br />
<div class="mainbody mainbody5" id="label_5"><br />
<p class="atop"><br />
<span>Reference</span><br />
</p><br />
<p class="first"><br />
[1]<a href="http://www.Brenda-enzymes.info/index.php"> http://www.Brenda-enzymes.info/index.php </a><br/><br />
[2] Gary D. Colby and Jiann-Shin Chen. Purification and Properties of 3-Hydroxybutyryl-Coenzyme A Dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Applied And Environmental Microbiology, Oct. 1992, p. 3297-3302<br/><br />
[3] Robert M. Waterson, Francis J. Castellino, G.Michael Hass and Robert L. Hill, Purification and Characterization of. Crotonase from Clostridium acetobutylicum, J. Biol. Chem. 1972, 247:5266-5271.<br/><br />
[4] Michel Rigoulet,1 Hugo Aguilaniu,1,3 Nicole Avéret,1 Odile Bunoust,1 Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae<br/><br />
[5] <a href="https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory">https://2012.igem.org/Team:Shenzhen/Result/YAO.Factory</a><br/><br />
[6] RUN-TAO YAN AND JIANN-SHIN CHEN Coenzyme A-Acylating Aldehyde Dehydrogenase from Clostridium beijerinckii NRRL B592<br/><br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html></div>Soipanhttp://2014.igem.org/File:Modeling001.pngFile:Modeling001.png2014-10-17T23:29:25Z<p>Soipan: </p>
<hr />
<div></div>Soipanhttp://2014.igem.org/Team:SCUT/Team/AttributionsTeam:SCUT/Team/Attributions2014-10-17T22:19:16Z<p>Soipan: </p>
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</div><br />
<div class="navibody navibody1"><br />
<p>Members>>></p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Notebook"><img src="https://static.igem.org/mediawiki/2014/6/65/Team2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introdution</p><br />
<p>CO<sub>2</sub> fixed simulation lab </p><br />
<p>Butanol lab notes</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Attributions"><img src="https://static.igem.org/mediawiki/2014/0/0e/Team3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Attributions>>></p><br />
</div><br />
<div class="navihead navihead4"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Acknowledgement"><img src="https://static.igem.org/mediawiki/2014/6/6f/Team4-01.png"></a><br />
</div><br />
<div class="navibody navibody4"><br />
<p>Acknowledgements>>></p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Our_University"><img src="https://static.igem.org/mediawiki/2014/3/3b/Team5-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>SCUT</p><br />
<p>Our University</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Attributions</span><br />
</p><br />
<p class="first"><br />
<span class="xiaobiaoti">Zhang Junjie </span><br/><br/><br />
the leader of SCUT iGEM team, was responsible for constructing and managing the team, instructing team members to design and implement the project. He was also in charge of contacting with professors and seniors for us, and supervised the progress of all parts and tasks. <br />
</p><br />
<p><br />
<span class="xiaobiaoti">Butanol Part</span><br/><br/><br />
Fan Chuyao, Hu WeiPeng were involved in butanol part and made great contribution to constructing and optimizing the butanol pathway in inter-mitochondrial and cytoplasm.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">CO<sub>2</sub> Fixation Part</span><br/><br/><br />
Peng Ye, Chen Zhanru, Yang Bina managed RuBisCo part. Peng Ye was in charge of PRK, Rubisco and CA enzyme, Wang Zhizheng also made efforts to assist Peng Ye. Chen ZhanRu made brilliant contributions and devote himself to testing the scaffold protein using fluorescent protein fused with ligand and he also helped Yang to construct the pathway of scaffold protein. Meanwhile he assisted design group in drawing some vivid pictures. As for Yang Bina, she was diligent and she made great efforts to construct different scaffold protein combinations in order to help to find the best one. She has created 32 biobricks in total.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Leading Peptide Part</span><br/><br/><br />
Li LinZhou, Zhao Yaran made efforts on leading peptide of subcellular. From searching to experiment planning, they have done all of necessary things. And they really cooperated very well during the whole summer vacation.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Wiki and Logo Design Group</span><br/><br/><br />
Wang Xiuyuan, Ling ZhangYan, and Pan Xueman have undertaken all the tasks of designing of the whole team. We did a lot work including logo, ppt templates, wiki and uniforms.Our group worked industriously on WIKI, with PPT as an onset. Taking step by step, as a result of clarified division and efficient cooperation of, they sophisticatedly design uniforms and posters for the IGEM.Wang feel honored to become one of the IGEMers, in this team, like he said :"I can not only show my talents for editing skills as well as contribute my ideas on design, but also meet with gifted and competent peers here. I feel good to strive for the same goal together."<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Modelling Group</span><br/><br/><br />
Huang Xiaobin, Wu Yuhang and Zhou Shiyan devoted their passion to modeling metabolic pathway to instruct our experiments. This group has a diversified background since the three members come from three different schools. Xiaobin majors in computer science, Yuhang majors in Biotech while Shiyan studies mathematics. Thus they provided more professional works for SCUT iGEM team.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Human Practice</span><br/><br/><br />
The Human Practice part of this year is completely in the charge of Wang Zhizheng. He has determined the topic of HP, “Synthetic Biology Beautifies Our Life”. He designed a variety of methods to promote this topic and make knowledge of synthetic biology and iGEM. He has opened the internet communication platform, compiled our original magazine,”iGEM Life”, traveled to HongKong, Shenzhen, Wuhan and some places to communicate with other iGEM team. What’s more, he and his assistants also did two surveys to observe the change of their attitude towards synthetic biology and iGEM. So he made considerable efforts and contributions to making HP part better.<br />
</p><br />
</div><br />
<br />
<br />
</body><br />
<br />
<br />
<br />
<br />
</html></div>Soipanhttp://2014.igem.org/Team:SCUT/Team/AttributionsTeam:SCUT/Team/Attributions2014-10-17T22:17:51Z<p>Soipan: </p>
<hr />
<div>{{Template:Team:SCUT/mainhead}}<br />
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<br />
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<div class="navihead navihead1"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Members"><img src="https://static.igem.org/mediawiki/2014/c/c6/Team1-01.png"></a><br />
</div><br />
<div class="navibody navibody1"><br />
<p>Members>>></p><br />
</div><br />
<div class="navihead navihead2"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Notebook"><img src="https://static.igem.org/mediawiki/2014/6/65/Team2-01.png"></a><br />
</div><br />
<div class="navibody navibody2"><br />
<p>Introdution</p><br />
<p>CO<sub>2</sub> fixed simulation lab </p><br />
<p>Butanol lab notes</p><br />
</div><br />
<div class="navihead navihead3"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Attributions"><img src="https://static.igem.org/mediawiki/2014/0/0e/Team3-01.png"></a><br />
</div><br />
<div class="navibody navibody3" id="show"><br />
<p onclick="scroll_1()">Attributions>>></p><br />
</div><br />
<div class="navihead navihead4"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Acknowledgement"><img src="https://static.igem.org/mediawiki/2014/6/6f/Team4-01.png"></a><br />
</div><br />
<div class="navibody navibody4"><br />
<p>Acknowledgements>>></p><br />
</div><br />
<div class="navihead navihead5"><br />
<a href="https://2014.igem.org/Team:SCUT/Team/Our_University"><img src="https://static.igem.org/mediawiki/2014/3/3b/Team5-01.png"></a><br />
</div><br />
<div class="navibody navibody5"><br />
<p>SCUT</p><br />
<p>Our University</p><br />
</div><br />
</div><br />
<br />
<div class="mainbody mainbody1" id="label_1"><br />
<p class="atop"><br />
<span>Attributions</span><br />
</p><br />
<p class="first"><br />
<span class="xiaobiaoti">Zhang Junjie </span><br/><br/><br />
the leader of SCUT iGEM team, was responsible for constructing and managing the team, instructing team members to design and implement the project. He was also in charge of contacting with professors and seniors for us, and supervised the progress of all parts and tasks. <br />
</p><br />
<p><br />
<span class="xiaobiaoti">Butanol Part</span><br/><br/><br />
Fan Chuyao, Hu WeiPeng were involved in butanol part and made great contribution to constructing and optimizing the butanol pathway in inter-mitochondrial and cytoplasm.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Co2 Fixation Part</span><br/><br/><br />
Peng Ye, Chen Zhanru, Yang Bina managed RuBisCo part. Peng Ye was in charge of PRK, Rubisco and CA enzyme, Wang Zhizheng also made efforts to assist Peng Ye. Chen ZhanRu made brilliant contributions and devote himself to testing the scaffold protein using fluorescent protein fused with ligand and he also helped Yang to construct the pathway of scaffold protein. Meanwhile he assisted design group in drawing some vivid pictures. As for Yang Bina, she was diligent and she made great efforts to construct different scaffold protein combinations in order to help to find the best one. She has created 32 biobricks in total.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Leading Peptide Part</span><br/><br/><br />
Li LinZhou, Zhao Yaran made efforts on leading peptide of subcellular. From searching to experiment planning, they have done all of necessary things. And they really cooperated very well during the whole summer vacation.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Wiki and Logo Design Group</span><br/><br/><br />
Wang Xiuyuan, Ling ZhangYan, and Pan Xueman have undertaken all the tasks of designing of the whole team. We did a lot work including logo, ppt templates, wiki and uniforms.Our group worked industriously on WIKI, with PPT as an onset. Taking step by step, as a result of clarified division and efficient cooperation of, they sophisticatedly design uniforms and posters for the IGEM.Wang feel honored to become one of the IGEMers, in this team, like he said :"I can not only show my talents for editing skills as well as contribute my ideas on design, but also meet with gifted and competent peers here. I feel good to strive for the same goal together."<br />
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<span class="xiaobiaoti">Modelling Group</span><br/><br/><br />
Huang Xiaobin, Wu Yuhang and Zhou Shiyan devoted their passion to modeling metabolic pathway to instruct our experiments. This group has a diversified background since the three members come from three different schools. Xiaobin majors in computer science, Yuhang majors in Biotech while Shiyan studies mathematics. Thus they provided more professional works for SCUT iGEM team.<br />
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<span class="xiaobiaoti">Human Practice</span><br/><br/><br />
The Human Practice part of this year is completely in the charge of Wang Zhizheng. He has determined the topic of HP, “Synthetic Biology Beautifies Our Life”. He designed a variety of methods to promote this topic and make knowledge of synthetic biology and iGEM. He has opened the internet communication platform, compiled our original magazine,”iGEM Life”, traveled to HongKong, Shenzhen, Wuhan and some places to communicate with other iGEM team. What’s more, he and his assistants also did two surveys to observe the change of their attitude towards synthetic biology and iGEM. So he made considerable efforts and contributions to making HP part better.<br />
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<p>Introdution</p><br />
<p>CO2 fixed simulation lab </p><br />
<p>Butanol lab notes</p><br />
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<p>Acknowledgements>>></p><br />
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<p>SCUT</p><br />
<p>Our University</p><br />
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<span>Attributions</span><br />
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<span class="xiaobiaoti">Zhang Junjie </span><br/><br/><br />
the leader of SCUT iGEM team, was responsible for constructing and managing the team, instructing team members to design and implement the project. He was also in charge of contacting with professors and seniors for us, and supervised the progress of all parts and tasks. <br />
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<p><br />
<span class="xiaobiaoti">Butanol Part</span><br/><br/><br />
Fan Chuyao, Hu WeiPeng were involved in butanol part and made great contribution to constructing and optimizing the butanol pathway in inter-mitochondrial and cytoplasm.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Co2 Fixation Part</span><br/><br/><br />
Peng Ye, Chen Zhanru, Yang Bina managed RuBisCo part. Peng Ye was in charge of PRK, Rubisco and CA enzyme, Wang Zhizheng also made efforts to assist Peng Ye. Chen ZhanRu made brilliant contributions and devote himself to testing the scaffold protein using fluorescent protein fused with ligand and he also helped Yang to construct the pathway of scaffold protein. Meanwhile he assisted design group in drawing some vivid pictures. As for Yang Bina, she was diligent and she made great efforts to construct different scaffold protein combinations in order to help to find the best one. She has created 32 biobricks in total.<br />
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<span class="xiaobiaoti">Leading Peptide Part</span><br/><br/><br />
Li LinZhou, Zhao Yaran made efforts on leading peptide of subcellular. From searching to experiment planning, they have done all of necessary things. And they really cooperated very well during the whole summer vacation.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Wiki and Logo Design Group</span><br/><br/><br />
Wang Xiuyuan, Ling ZhangYan, and Pan Xueman have undertaken all the tasks of designing of the whole team. We did a lot work including logo, ppt templates, wiki and uniforms.Our group worked industriously on WIKI, with PPT as an onset. Taking step by step, as a result of clarified division and efficient cooperation of, they sophisticatedly design uniforms and posters for the IGEM.Wang feel honored to become one of the IGEMers, in this team, like he said :"I can not only show my talents for editing skills as well as contribute my ideas on design, but also meet with gifted and competent peers here. I feel good to strive for the same goal together."<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Modelling Group</span><br/><br/><br />
Huang Xiaobin, Wu Yuhang and Zhou Shiyan devoted their passion to modeling metabolic pathway to instruct our experiments. This group has a diversified background since the three members come from three different schools. Xiaobin majors in computer science, Yuhang majors in Biotech while Shiyan studies mathematics. Thus they provided more professional works for SCUT iGEM team.<br />
</p><br />
<p><br />
<span class="xiaobiaoti">Human Practice</span><br/><br/><br />
The Human Practice part of this year is completely in the charge of Wang Zhizheng. He has determined the topic of HP, “Synthetic Biology Beautifies Our Life”. He designed a variety of methods to promote this topic and make knowledge of synthetic biology and iGEM. He has opened the internet communication platform, compiled our original magazine,”iGEM Life”, traveled to HongKong, Shenzhen, Wuhan and some places to communicate with other iGEM team. What’s more, he and his assistants also did two surveys to observe the change of their attitude towards synthetic biology and iGEM. So he made considerable efforts and contributions to making HP part better.<br />
</p><br />
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