Difference between revisions of "Team:NPU-China/CompositeParts"

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         <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/9/95/NPU%E9%A2%98%E7%9B%AE%E9%80%9A%E6%A0%8Fbasicparts.jpg">
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         <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/6/6b/%E9%A2%98%E7%9B%AE%E9%80%9A%E6%A0%8Fbasicparts.jpg">
 
         <div class="container" style="padding-top:70px">
 
         <div class="container" style="padding-top:70px">
 
             <div class="row">
 
             <div class="row">
 
                 <div class="col-md-12">
 
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−
 
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                    <div class="col-lg-10 col-centered">
 
 
                     <p>
 
                     <p>
−
                    <table class="table table-bordered table-hover" style="background-color:#FFF;">
+
                        <table class="table table-bordered table-hover" style="background-color:#FFF;">
−
                    <thead>
+
                            <thead>
−
                            <tr>
+
                                <tr>
−
                                <th>Name</th>
+
                                    <th>Name</th>
−
                                <th>Type</th>
+
                                    <th>Type</th>
−
                                <th>Description</th>
+
                                    <th>Description</th>
−
                                <th>Length</th>
+
                                    <th>Length</th>
−
                            </tr>
+
                                </tr>
−
                        </thead>
+
                            </thead>
−
                        <tbody>
+
                            <tbody>
−
                            <tr>
+
                                <tr>
−
                                <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347005">BBa_K2347005</a></td>
+
                                    <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347000">BBa_K2347000</a></td>
−
                                <td>composite</td>     
+
                                    <td>Coding</td>     
−
                                <td>DHAP/G3P->acrylic acid</td>
+
                                    <td>DHAP/G3P->acrylic acid</td>
−
                                <td>2862bp</td>         
+
                                    <td>1719bp</td>         
−
                            </tr>
+
                                </tr>
−
                            <tr>
+
                                <tr>
−
                                <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347006">BBa_K2347006</a></td>
+
                                    <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347001">BBa_K2347001</a></td>
−
                                <td>composite</td>     
+
                                    <td>Coding</td>     
−
                                <td>glycerol->DHA</td>
+
                                    <td>glycerol->DHA</td>
−
                                <td>2862bp</td>         
+
                                    <td>1113bp</td>         
−
                            </tr>
+
                                </tr>
−
                            <tr>
+
                                <tr>
−
                                <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347007">BBa_K2347007</a></td>
+
                                    <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347002">BBa_K2347002</a></td>
−
                                <td>composite</td>     
+
                                    <td>Coding</td>     
−
                                <td>DHA->DHAP</td>
+
                                    <td>DHA->DHAP</td>
−
                                <td>2862bp</td>         
+
                                    <td>1827bp</td>         
−
                            </tr>
+
                                </tr>
−
                            <tr>
+
                                <tr>
−
                                <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347008">BBa_K2347008</a></td>
+
                                    <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347003">BBa_K2347003</a></td>
−
                                <td>composite</td>     
+
                                    <td>Coding</td>     
−
                                 <td>NADH->NAD+</td>
+
                                    <td>H2O2->O2</td>
−
                                <td>2862bp</td>         
+
                                    <td>1584bp</td>       
−
                            </tr>
+
                                 </tr>
−
                    </tbody>
+
<tr>
−
                    </table>
+
                                    <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K2347004">BBa_K2347004</a></td>
−
<h4>
+
                                    <td>Coding</td>   
 +
                                    <td>NADH->NAD+</td>
 +
                                    <td>618bp</td>         
 +
                                </tr>
 +
                            </tbody>
 +
                        </table>
 +
<h2 style="text-align:center"> Our best basic part, ceaS2</h2>
 +
                    <h4>CEAS, which is N2- (2-carboxyethyl) arginine synthase, is an enzyme in Streptomyces clavuligerus, EC
 +
                        2.5.1.66. CEAS is a TPP-related enzyme, can catalyze the condensation of D-G3P and L-Arg with the
 +
                        involvement of TPP and magnesium ions (thiamine pyrophosphate) to produce N2- (2-carboxyethyl) arginine,
 +
                        which will continue to participate in the biosynthesis of clavulanic acid as the first intermediate.<br>
 +
                        <center><img src="https://static.igem.org/mediawiki/2017/a/ac/Ceas2.png" class="img-responsive"></center>
 +
                     
 +
                        <br>According to the earlier literature, CEAS (N2-(2-carboxyethyl) arginine synthase) was a synergistic
 +
                        effect of Ceas1 and Ceas, namely, N2- (2-carboxyethyl) arginine came from the condensation of G3P
 +
                        and L-Arg, which was catalyzed by Ceas1 and Ceas2. The process would be accompanied by the formation
 +
                        of acrylic acid[1]. But with the deepening of the study, Matthew E.C. Caines found that Ceas2 played
 +
                        the main role in the catalytic process. And they speculated the catalytic mechanism of Ceas2, as
 +
                        shown below [2].</h4>
 +
                        <center><img src="https://static.igem.org/mediawiki/2017/2/29/Basicpart.png" class="img-responsive"></center>
  
−
              <div class="col-md-12" style="padding-top:30px">
+
                        <h4>
−
                     <div class="col-md-6">
+
                    <h2 style="text-align:center"> How we use these basic parts?</h2>
−
<center><h4>1.pSBC3-ADH1+gld+tGPD1<br><h4></center>
+
                     <center> <img src="https://static.igem.org/mediawiki/2017/c/ce/5%2C6%2C7%2C8%EF%BC%8C10.png" class="img-responsive"></center>
−
                  <center> <h4>In this part, the promoter of gld is ADH1 promoter, and the terminator is tGPD1 terminator. ADH1 promoter is
+
                    <center>Fig.5:DAK;6:NOX;7,ceaS2;8:gld;10:CAT</center>
−
                        a kind of promoter which has strong expression and tGPD1 terminator is a terminator with rather strong
+
                    <h4>We designed a GDC (GlyDH-DAK-Ceas2) pathway that could produce acrylic acid using glycerol. In this pathway,  
−
                        expression ability. So they can both increase the expression of genes. At the same time they are constitutive
+
                    GlyDH (Glycerol dehydrogenase，BBa_K2347001) can efficiently convert glycerol to DHA (1,3-Dihydroxyacetone), and  
−
                        promoters and terminators, which have the ability to integrate gene fragment, gld, into the chromosome of  
+
                    then DAK（Dihydroxyacetone kinase，BBa_K2347002） enzyme can phosphorylate  DHA to DHAP, and then the ceaS2 Enzyme
−
                        yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part
+
                    BBa_K2347000）can make it into acrylic acid.
−
                        pSBC3-PGK1 + DAK + TPFK1 and the YCplac33 plasmid vector to form intact plasmid with URA deficient.
+
                    <br>In addition, since GlyDH is  NAD + dependent,  in order to increase the supply of reducing force, we also
−
                        </h4><br></center>
+
                    added the NOX (NADH dehydrogenase, BBa_K2347003) and CAT (Catalase, BBa_K2347004) to this pathway, providing
−
                      <center> <img src="https://static.igem.org/mediawiki/2017/e/e5/NPU-pSB1C3-ADH1-GlyDH-tGPD1.png" class="img-responsive"></center>
+
                    the reduction force for GlyDH through two layers of substrate.  Finally, we created the new combination of GNCDC
−
                         <h4> </h4>
+
                    (GlyDH-NOX-CAT-DAK-ceaS2) pathway using five enzymes from BBa_K2347000 to BBa_K2347004 to produce acrylic acid.</h4>
 +
                        <br>
 +
                      <center><img src="https://static.igem.org/mediawiki/2017/1/1b/Basicpart2.png" class="img-responsive"></center>
 +
                         <br>
 +
                    <h5>[1] MERSKI M, TOWNSEND C A. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of
 +
                        Clavulanic Acid Biosynthesis [J]. Journal of the American Chemical Society, 2007, 129(51): 15750-1.
 +
                        <br> [2] CAINES M E, ELKINS J M, HEWITSON K S, et al. Crystal structure and mechanistic implications
 +
                        of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway
 +
                        [J]. Journal of Biological Chemistry, 2004, 279(7): 5685-92.</h5>
 +
                        </h4>
 
                     </div>
 
                     </div>
−
 
+
<div class="col-md-3">
−
 
+
                        
−
                    <div class="col-md-6">
+
−
 
+
−
  <center> <h4>2.pSBC3-PGK1+DAK+TPFK1<br><h4></center>
+
−
                  <center>  <h4>In this part, the promoter of DAK is PGK1 promoter, and the terminator is TPFK1 terminator. PGK1 promoter 
+
−
                        is a kind of promoter which has strong expression and TPFK1 terminator is a terminator with rather strong
+
−
                        expression ability. So they can both increase the expression of genes. At the same time they are constitutive 
+
−
                        promoters and terminators, which have the ability to integrate gene fragment, DAK, into the chromosome
+
−
                        of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part
+
−
                        pSBC3-ADH1+gld+tGPD1 and the YCplac33 plasmid vector to form intact plasmid with URA deficient.
+
−
                        </h4><br></center>
+
−
 
+
−
                       <center>  <img src="https://static.igem.org/mediawiki/2017/e/e8/PSB1C3-PGK1-DAK-tPFK1.png" class="img-responsive"></center>
+
 
                         <h4> </h4>
 
                         <h4> </h4>
 
                     </div>
 
                     </div>
−
 
 
                 </div>
 
                 </div>
−
  <center>  <h4>pSBC3-ADH1+gld+tGPD1 & pSBC3-PGK1+DAK+TPFK1<br><h4></center>
 
−
                    <h4>The enzyme GlyDH (gld) and DAK can be efficiently expressed in the yeast cell, and the gene of the
 
−
enzyme GlyDH (gld) and DAK are integrated into the chromosome. In this pathway, GlyDH (Glycerol
 
−
dehydrogenase) can efficiently convert glycerol to DHA (1,3-Dihydroxyacetone), and then DAK
 
−
(Dihydroxyacetone kinase) phosphorylate DHA into DHAP, and then DHAP is catalyzed into acrylic acid
 
−
by ceaS2.
 
−
                        </h4>
 
−
<center>  <img src="https://static.igem.org/mediawiki/2017/6/65/YCplac33-URA-gld-DAK.png" class="img-responsive"></center>
 
  
−
</h4>
 
−
              <h4>3.pSBC3-pTDH3+ceas2+tPFK1<br><h4>
 
−
                    <h4>In this part, the promoter of ceaS2 is pTDH3 promoter, and the terminator is tPFK1 terminator. pTDH3 
 
−
                        promoter is a kind of promoter which has strong expression and tPFK1 terminator is a terminator with
 
−
                        rather strong expression ability. So they can both increase the expression of genes. At the same time they 
 
−
                        are constitutive promoters and terminators, which have the ability to integrate gene fragment, ceaS2, into
 
−
                        the chromosome of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part 
 
−
                        with the YCplac33 plasmid vector to form intact plasmid with LEU deficient.
 
−
                        </h4><br>
 
−
<h4>
 
−
              <div class="col-md-12" style="padding-top:30px">
 
−
                    <div class="col-md-6">
 
−
                        <img src="https://static.igem.org/mediawiki/2017/6/6c/PSB1C3-pTDH3%2Bceas2%2BtPFK1.png" class="img-responsive">
 
−
                        <h4> </h4>
 
 
                     </div>
 
                     </div>
−
                    <div class="col-md-6">
 
−
                        <img src="https://static.igem.org/mediawiki/2017/0/06/YCplac-pTDH3-ceaS2-tPFK1.png" class="img-responsive">
 
−
                        <h4> </h4>
 
−
                   
 
−
                </div>
 
−
</div>
 
  
  
−
 
−
  <h4>In the yeast cells, the enzyme ceaS2 can be efficiently expressed and the gene of ceas2 is integrated into
 
−
the chromosome. With the help of TPP (Thiamine pyrophosphate) and magnesium ions, ceaS2 can
 
−
catalyze the production of acrylic acid with DHAP (dihydroxy acetone phosphate) and G3P
 
−
(glyceraldehyde 3-phosphate ) as substrate. </h4><br>
 
−
 
−
</h4>
 
−
 
−
 
−
                    <h4>4.pSBC3-TEF2+NOX+tRPS2<br><h4>
 
−
                    <h4>In this part, the promoter of NOX is TEF2 promoter, and the terminator is tRPS2 terminator. TEF2 promoter is
 
−
a kind of promoter which has strong expression and tRPS2 terminator is a terminator with rather strong
 
−
expression ability. So they can both increase the expression of genes. At the same time they are constitutive
 
−
promoters and terminators, which have the ability to integrate gene fragment, NOX, into the chromosome
 
−
of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part
 
−
pSBC3-pTDH3+ceas2+tPFK1and the YCplac33 plasmid vector to form intact plasmid with LEU deficient.
 
−
                        </h4><br>
 
−
        <h4>        <div class="col-md-12" style="padding-top:30px">
 
−
                    <div class="col-md-6">
 
−
                        <img src="https://static.igem.org/mediawiki/2017/c/c4/PSB1C3-TEF2-NOX-tRPS2.png" class="img-responsive">
 
−
                        <h4> </h4>
 
−
                    </div>
 
−
                    <div class="col-md-6">
 
−
                        <img src="https://static.igem.org/mediawiki/2017/8/86/YCplac33-Leu-NOX-ceaS2.png" class="img-responsive">
 
−
                        <h4> </h4>
 
−
                   
 
−
                </div>
 
−
</div>
 
−
                    <h4>It can efficiently express the enzyme NOX in yeast and integrate NOX into the chromosome. Since GlyDH
 
−
is an NAD+ -dependent enzyme, NOX and CAT(which already exists in yeast) provide the required
 
−
reduction force for GLYDH through the two layers of substrate level cycle.
 
−
                        </h4><br>
 
−
  </h4>
 
 
                 </div>
 
                 </div>
  

Revision as of 02:33, 2 November 2017

Name Type Description Length
BBa_K2347000 Coding DHAP/G3P->acrylic acid 1719bp
BBa_K2347001 Coding glycerol->DHA 1113bp
BBa_K2347002 Coding DHA->DHAP 1827bp
BBa_K2347003 Coding H2O2->O2 1584bp
BBa_K2347004 Coding NADH->NAD+ 618bp

Our best basic part, ceaS2

CEAS, which is N2- (2-carboxyethyl) arginine synthase, is an enzyme in Streptomyces clavuligerus, EC 2.5.1.66. CEAS is a TPP-related enzyme, can catalyze the condensation of D-G3P and L-Arg with the involvement of TPP and magnesium ions (thiamine pyrophosphate) to produce N2- (2-carboxyethyl) arginine, which will continue to participate in the biosynthesis of clavulanic acid as the first intermediate.

According to the earlier literature, CEAS (N2-(2-carboxyethyl) arginine synthase) was a synergistic effect of Ceas1 and Ceas, namely, N2- (2-carboxyethyl) arginine came from the condensation of G3P and L-Arg, which was catalyzed by Ceas1 and Ceas2. The process would be accompanied by the formation of acrylic acid[1]. But with the deepening of the study, Matthew E.C. Caines found that Ceas2 played the main role in the catalytic process. And they speculated the catalytic mechanism of Ceas2, as shown below [2].

How we use these basic parts?

Fig.5:DAK;6:NOX;7,ceaS2;8:gld;10:CAT

We designed a GDC (GlyDH-DAK-Ceas2) pathway that could produce acrylic acid using glycerol. In this pathway, GlyDH (Glycerol dehydrogenase，BBa_K2347001) can efficiently convert glycerol to DHA (1,3-Dihydroxyacetone), and then DAK（Dihydroxyacetone kinase，BBa_K2347002） enzyme can phosphorylate DHA to DHAP, and then the ceaS2 Enzyme BBa_K2347000）can make it into acrylic acid.
In addition, since GlyDH is NAD + dependent, in order to increase the supply of reducing force, we also added the NOX (NADH dehydrogenase, BBa_K2347003) and CAT (Catalase, BBa_K2347004) to this pathway, providing the reduction force for GlyDH through two layers of substrate. Finally, we created the new combination of GNCDC (GlyDH-NOX-CAT-DAK-ceaS2) pathway using five enzymes from BBa_K2347000 to BBa_K2347004 to produce acrylic acid.



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