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

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                     <h4>CEAS, which is N2- (2-carboxyethyl) arginine synthase, is an enzyme in Streptomyces clavuligerus, EC
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                <h2>Composite part<h2><br>
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                         2.5.1.66. CEAS is a TPP-related enzyme, can catalyze the condensation of D-G3P and L-Arg with the
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                     <h4>1.1pSBC3-ADH1+gld+tGPD1<br><h4>
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                         involvement of TPP and magnesium ions (thiamine pyrophosphate) to produce N2- (2-carboxyethyl) arginine,
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                    <h4>In this part, the promoter of gld is ADH1 promoter, the terminator is tGPD1 terminator. ADH1 promoter is
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                         which will continue to participate in the biosynthesis of clavulanic acid as the first intermediate.
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                         a kind of promoter which has strong expression and tGPD1 terminator is a terminator with rather strong
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                         According to the earlier literature, CEAS (N2-(2-carboxyethyl) arginine synthase) was a synergistic
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                        expression ability. So they can both increase the expression of genes. At the same time they are constitutive
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                         effect of Ceas1 and Ceas, namely, N2- (2-carboxyethyl) arginine came from the condensation of G3P
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                        promoters and terminators, which have the ability to integrate gene fragment, gld, into the chromosome of
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                         and L-Arg, which was catalyzed by Ceas1 and Ceas2. The process would be accompanied by the formation
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                         yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part
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                         of acrylic acid[1]. But with the deepening of the study, Matthew E.C. Caines found that Ceas2 played
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                        pSBC3-PGK1 + DAK + TPFK1 and the YCplac33 plasmid vector to form intact plasmid with URA deficient.
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                         the main role in the catalytic process. And they speculated the catalytic mechanism of Ceas2, as
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                        </h4><br>
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                         shown below [2].</h4>
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                    <h4>2.pSBC3-PGK1+DAK+TPFK1<br><h4>
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                        <br>
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                    <h4>In this part, the promoter of DAK is PGK1 promoter, the terminator is TPFK1 terminator. PGK1 promoter 
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                     <div align="center">
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                         is a kind of promoter which has strong expression and TPFK1 terminator is a terminator with rather strong
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                        <img src="https://static.igem.org/mediawiki/2017/2/29/Basicpart.png" class="img-responsive">
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                        expression ability. So they can both increase the expression of genes. At the same time they are constitutive 
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                         <br><br><br>
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                         promoters and terminators, which have the ability to integrate gene fragment, DAK, into the chromosome
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                        <img src="https://static.igem.org/mediawiki/2017/1/1b/Basicpart2.png" class="img-responsive">
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                         of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part
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                         <br>
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                         pSBC3-ADH1+gld+tGPD1 and the YCplac33 plasmid vector to form intact plasmid with URA deficient.
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                     </div>
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                         </h4><br>
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                    <h4>3.pSBC3-pTDH3+ceas2+tPFK1<br><h4>
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                     <h5>[1] MERSKI M, TOWNSEND C A. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of
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                    <h4>In this part, the promoter of ceaS2 is pTDH3 promoter, the terminator is tPFK1 terminator. pTDH3 
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                        Clavulanic Acid Biosynthesis [J]. Journal of the American Chemical Society, 2007, 129(51): 15750-1.
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                         promoter is a kind of promoter which has strong expression and tPFK1 terminator is a terminator with
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                         <br> [2] CAINES M E, ELKINS J M, HEWITSON K S, et al. Crystal structure and mechanistic implications
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                        rather strong expression ability. So they can both increase the expression of genes. At the same time they  
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                        of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway
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                        are constitutive promoters and terminators, which have the ability to integrate gene fragment, ceaS2, into
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                        [J]. Journal of Biological Chemistry, 2004, 279(7): 5685-92.</h5>
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                        the chromosome of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part 
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                         with the YCplac33 plasmid vector to form intact plasmid with LEU deficient.
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                        </h4><br>
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                     <h4>4.pSBC3-TEF2+NOX+tRPS2<br><h4>
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                    <h4>In this part, the promoter of NOX is TEF2 promoter, 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>
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                    <h4>4.pSBC3-TEF2+NOX+tRPS2<br><h4>
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                    <h4>In this part, the promoter of NOX is TEF2 promoter, 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>
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                     <h3>How we use composite parts?<h3><br>  
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                     <h4>1.pSBC3-pTDH3+ceas2+tPFK1<br><h4>
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                    <h4>In the yeast cells, the enzyme ceaS2 can be efficiently expressed and the gene of ceas2 is integrated into
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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
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(glyceraldehyde 3-phosphate ) as substrate.
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                         </h4><br>
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                    <h4>2.pSBC3-ADH1+gld+tGPD1和pSBC3-PGK1+DAK+TPFK1<br><h4>
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                    <h4>The enzyme GlyDH (gld) and DAK can be efficiently expressed in the yeast cell, and the gene of the
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enzyme GlyDH (gld) and DAK are integrated into the chromosome. In this pathway, GlyDH (Glycerol
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dehydrogenase) can efficiently convert glycerol to DHA (1,3-Dihydroxyacetone), and then DAK
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(Dihydroxyacetone kinase) phosphorylate DHA into DHAP, and then DHAP is catalyzed into acrylic acid
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by ceaS2.
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                        </h4><br>
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                    <h4>3.pSBC3-TEF2+NOX+tRPS2<br><h4>
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                    <h4>It can efficiently express the enzyme NOX in yeast and integrate NOX into the chromosome. Since GlyDH
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is an NAD+ -dependent enzyme, NOX and CAT(which already exists in yeast) provide the required
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reduction force for GLYDH through the two layers of substrate level cycle.
 +
                        </h4><br>
 
                 </div>
 
                 </div>
  

Revision as of 14:38, 1 November 2017

Composite part


1.1pSBC3-ADH1+gld+tGPD1

In this part, the promoter of gld is ADH1 promoter, the terminator is tGPD1 terminator. ADH1 promoter is a kind of promoter which has strong expression and tGPD1 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, gld, into the chromosome of yeast, thereby reducing the burden of plasmid expression in yeast. We linked this part with part pSBC3-PGK1 + DAK + TPFK1 and the YCplac33 plasmid vector to form intact plasmid with URA deficient.


2.pSBC3-PGK1+DAK+TPFK1

In this part, the promoter of DAK is PGK1 promoter, 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.


3.pSBC3-pTDH3+ceas2+tPFK1

In this part, the promoter of ceaS2 is pTDH3 promoter, 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.


4.pSBC3-TEF2+NOX+tRPS2

In this part, the promoter of NOX is TEF2 promoter, 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.


4.pSBC3-TEF2+NOX+tRPS2

In this part, the promoter of NOX is TEF2 promoter, 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.


How we use composite parts?


1.pSBC3-pTDH3+ceas2+tPFK1

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.


2.pSBC3-ADH1+gld+tGPD1和pSBC3-PGK1+DAK+TPFK1

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.


3.pSBC3-TEF2+NOX+tRPS2

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.