Difference between revisions of "Team:Xiamen City/Contribution"

 
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             <!--Human Practice-->
 
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                 <a href="https://2021.igem.org/Team:Xiamen_City/Human_Practices">Human Practice</a>
 
                 <a href="https://2021.igem.org/Team:Xiamen_City/Human_Practices">Human Practice</a>
 
                 <div class="child-nav">
 
                 <div class="child-nav">
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                                 Practice</a>
 
                                 Practice</a>
 
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                         </li>
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                         <li class="child-item">
 
                             <a href="https://2021.igem.org/Team:Xiamen_City/Communication">Communication</a>
 
                             <a href="https://2021.igem.org/Team:Xiamen_City/Communication">Communication</a>
 
                         </li>
 
                         </li>
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             <!--Parts-->
 
             <!--Parts-->
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                 <a href="https://2021.igem.org/Team:Xiamen_City/Parts">Parts</a>
 
                 <a href="https://2021.igem.org/Team:Xiamen_City/Parts">Parts</a>
 
                 <div class="child-nav">
 
                 <div class="child-nav">
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                             <a href="https://2021.igem.org/Team:Xiamen_City/Engineering">Engineering</a>
 
                             <a href="https://2021.igem.org/Team:Xiamen_City/Engineering">Engineering</a>
 
                         </li>
 
                         </li>
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                         <li class="child-item">
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                         <li class="child-item active">
 
                             <a href="https://2021.igem.org/Team:Xiamen_City/Contribution">Contribution</a>
 
                             <a href="https://2021.igem.org/Team:Xiamen_City/Contribution">Contribution</a>
 
                         </li>
 
                         </li>
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         <div class="content-middle">
 
         <div class="content-middle">
 
             <section class="article p-t-30 p-b-54">
 
             <section class="article p-t-30 p-b-54">
−
                 <h1 class="content-header">Implementation</h1>
+
                 <h1 class="content-header">Contribution</h1>
  
 
                 <section>
 
                 <section>
−
                     <p>We introduced CRISPR technology and PCR technology to our community in a PowerPoint presentation and
+
                     <h1 class="title">BBa_K4002002</h1>
−
                        a recorded video. The majority of our educational audience were children. Therefore, we streamlined
+
−
                        the PowerPoint and video instruction to make it easier to understand. Initially, we included a
+
−
                        detailed description of CRISPR and PCR technologies in the PowerPoint. All of the materials
+
−
                        mentioned in the PPT are told with vivid cartoon and pictures. <span style="color: red">Here attached the educational
+
−
                    PPT.</span></p>
+
−
                    <img class="m-t-12" src="https://static.igem.org/mediawiki/2021/b/bc/T--Xiamen_City--img_communication_2.jpg" alt="" style="width: 70%;">
+
−
                    <video src="https://static.igem.org/mediawiki/2021/6/6d/T--Xiamen_City--video_communication.mp4"
+
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                          controls preload="metadata"
+
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                          class="m-t-24 m-b-12"
+
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                          style="width: 100%; height: 100%">
+
−
                    </video>
+
−
                    <p>In these material, we showed children process of yeast decomposition and the importance of
+
−
                        multifunctional yeast in the winemaking. We also explained gene-editing technology in the simplest
+
−
                        terms. We gave the children some knowledge about the structure of plant roots, stems and leaves.
+
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                        Dividing the children into groups, we organized the students to work together to put together the
+
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                        root and leaf structure of plants, which brought them a lot of joy. </p>
+
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                    <p>We have also set up our WeChat official account as an important communication channel with the
+
−
                        public. Initially, the account functions as a recorder of what we have done. As our followers
+
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                        increased rapidly, we found it a great means to educate the public about gene-editing technique. We
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                        have published 7 articles and some of them have been read by over 1000 people.</p>
+
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                    <img class="m-t-12" src="https://static.igem.org/mediawiki/2021/0/07/T--Xiamen_City--img_communication_3.jpg" alt="" style="width: 70%;">
+
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                    <img class="m-t-18" src="https://static.igem.org/mediawiki/2021/6/6c/T--Xiamen_City--img_communication_5.jpg" alt="" style="width: 70%;">
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                    <p>As we have rent an office to work together in a crowded office building, we took advantage of great
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                        human traffic in the morning and evening peak. We designed a roll up banner and placed it in front
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                        of our office, and took turns to hand out brochures with our official account's QR code of our
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                        official account. To avoid people's aversion, we also handed out free masks with our cute logo on
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                        it. By doing this, our official account obtained a great number of new followers.</p>
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                    <img class="m-t-12" src="https://static.igem.org/mediawiki/2021/2/29/T--Xiamen_City--img_communication_4.jpg" alt="" style="width: 50%;">
+
  
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                     <p>In order to enlarge our impact, we intend to make a MV for our team. We rewrote the lyric of STAY(
+
                    <h2 class="title2">== Profile BBa_K4002002==</h2>
−
                        from the Kid LAROI/Justin Bieber). We talked about our development procedures of designing
+
                     <p>Name: HXK1</p>
−
                        multifunctional yeast and our future market entrance plan. We are still designing and making the
+
                    <p>Base Pairs: 1458 bp</p>
−
                        final video. We plan to release the video on several popular platforms, such as YouTube, Bilibili
+
                    <p>Origin: <i>Saccharomyces cerevisiae</i>, genome</p>
−
                        and Weibo.</p>
+
                    <p>Properties: An enzyme catalyze the phosphorylation of hexose</p>
  
−
                     <br>
+
                     <h2 class="title2">== Usage and Biology ==</h2>
−
                     <p>Here attached the lyric ( we recompose the lyrics).</p>
+
                     <p>This is a sequence coding enzyme HXK1. This enzyme can catalyze the phosphorylation of hexose to
 +
                        hexose 6-phosphate (D-glucose 6-phosphate and D-fructose 6-phosphate, respectively). Besides, it can
 +
                        mediate the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose
 +
                        6-phosphate.</p>
 +
                </section>
  
−
                    <section style="white-space: pre-line">
+
                <section>
−
                        <section style="color: #008AC6">We do the same, thing we planned that we had to do.
+
                    <h1 class="title">BBa_K4002003</h1>
  
−
                            We tried our best, even if it's hard to do we
+
                    <h2 class="title2">== Profile ==</h2>
 +
                    <p>Name: pYES2-gRNA-hyg-MCS</p>
 +
                    <p>Base Pairs: 388 bp</p>
 +
                    <p>Origin: <i>From article, Addgene</i></p>
 +
                    <p>Properties: A piece of RNA complementary to the target gene.</p>
  
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                            never regret any decisions we have made
+
                    <h2 class="title2">== Usage and Biology ==</h2>
 +
                    <p>BBa_K4002003 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they
 +
                        form complexes with. And this sequence is inserted into plasmid vector.</p>
  
−
                            Cuz it worth it,it worth it, hey
+
                    <h2 class="title2">== Construct design ==</h2>
−
                         </section>
+
                    <p>sgRNA is inserted into plasmid vector. Plasmid sequence map is shown in Figure 1. </p>
−
                         I'm tired of, Wiki and Business plan
+
                    <div class="img-container m-t-12 m-b-12">
 +
                         <img src="https://static.igem.org/mediawiki/2021/7/74/T--Xiamen_City--img_contribution_1.jpg" alt="" style="width: 80%;">
 +
                         <span class="figure">Figure 1. Schematic map of pYES2-gRNA-hyg-MCS plasmid.</span>
 +
                    </div>
 +
                </section>
  
−
                        I realize the time that I wasted here
+
                <section>
 +
                    <h1 class="title">BBa_K4002004</h1>
  
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                        I feel like I can't feel the light of life
+
                    <h2 class="title2">== Profile ==</h2>
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                        <section style="color: #008AC6">
+
                    <p>Name: pHCas9-Nours</p>
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                            i.G.E.M. is what we're striving for.
+
                    <p>Base Pairs: 4837bp</p>
 +
                    <p>Origin: <i>Streptococcus pyogenes</i>, Addgene</p>
 +
                    <p>Properties: An endonuclease enzyme associated with the CRISPR.</p>
  
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
                    <h2 class="title2">== Usage and Biology ==</h2>
 +
                    <p>This is a sequence coding pHCas9 protein. This protein is a dual RNA-guided DNA endonuclease enzyme
 +
                        associated with the (CRISPR) adaptive immune system. The Cas9 protein has been heavily utilized as a
 +
                        genome engineering tool to induce site-directed double-strand breaks in DNA. The genes that encode
 +
                        the Cas9 protein and sgRNA were introduced into a cell and programmed to change its target gene.
 +
                        sgRNA has regions that are complementary to the target sequence. A complex consisting of sgRNA and
 +
                        Cas9 protein is formed inside the cell and binds to target sites.</p>
 +
                </section>
  
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
                <section>
 +
                    <h1 class="title">BBa_K4002005</h1>
  
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
                    <h2 class="title2">== Profile ==</h2>
 +
                    <p>Name: <i>endo-pgaA</i></p>
 +
                    <p>Base Pairs: 1113bp</p>
 +
                    <p>Origin: <i>Aspergillus niger</i> SC323, genome</p>
 +
                    <p>Properties: An enzyme degradation of pectin</p>
  
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                            i.G.E.M. is what we're striving for.
+
                    <h2 class="title2">==== Usage and Biology ====</h2>
 +
                    <p>Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic
 +
                        acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and
 +
                        poly-alpha-1,4-galacturonide glycanohydrolase. This part as a repair template DNA was connected with
 +
                        homology arm of HXK1.</p>
 +
                </section>
  
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                            We do the same, thing we planned that we had to do.
+
                <section>
 +
                    <h1 class="title">BBa_K4002006</h1>
  
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                            We tried our best, even if it's hard to do we
+
                    <h2 class="title2">== Profile ==</h2>
 +
                    <p>Name: HR-L-endo-pgaA-HR-R</p>
 +
                    <p>Base Pairs: 2135 bp</p>
 +
                    <p>Origin: Ssynthetic</p>
 +
                    <p>Properties: CRISPR technology repair template for build a type of multi-functional yeast</p>
  
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                            never regret any decisions we have made
+
                    <h2 class="title2">== Usage and Biology ==</h2>
 +
                    <p>Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic
 +
                        acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and
 +
                        poly-alpha-1,4-galacturonide glycanohydrolase. Pectin is a significant carbohydrate component that
 +
                        comprises plant cell walls. The brewer's yeast uses sugar in the fruit juice to produce alcohol, and
 +
                        pectinase destroys the pectin located in the cell wall to improve the juice yield and eliminate the
 +
                        cloudiness of the fruit wine. HR-L and HR-R are homology arm, refers to the flanking sequence on
 +
                        both sides of the HXK1.</p>
  
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                            Cuz it worth it,it worth it, hey
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                    <h2 class="title2">== Construct design ==</h2>
−
                        </section>
+
                    <p>HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the
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                         We are the pecteast and we work on that(Ooh)
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                         sequence of pgaA inserted in the homology arm (Figure 1 and 2). </p>
  
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                         It is the reason that we gathered round(Ooh)
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                    <div class="img-container m-t-12 m-b-12">
 +
                         <img src="https://static.igem.org/mediawiki/2021/2/24/T--Xiamen_City--img_contribution_2.jpg" alt="" style="width: 90%;">
 +
                        <span class="figure">Figure 1. HR-L-endo-pgaA-HR-R box.</span>
 +
                    </div>
  
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                        It's been difficult for us to trust (Ooh)
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                    <div class="img-container m-t-12 m-b-12">
−
 
+
                         <img src="https://static.igem.org/mediawiki/2021/8/87/T--Xiamen_City--img_contribution_3.jpg" alt="" style="width: 90%;">
−
                        That we've already finished all the stuffs(Ooh)
+
                        <span class="figure">Figure 2. Schematic map of HR-L-endo-pgaA-HR-R.</span>
−
 
+
                     </div>
−
                        Ain't no way that I can leave you pecteast
+
−
 
+
−
                        'Cause you ain't ever left me empty-handed
+
−
 
+
−
                        And you know that I know that I can't live without you
+
−
 
+
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                        So, pecteast, stay
+
−
                         <section style="color: #008AC6">
+
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
−
 
+
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
−
 
+
−
                            Oh, ooh-woah (Oh, ooh-woah)
+
−
 
+
−
                            i.G.E.M. is what we're striving for.
+
−
 
+
−
                            We do the same, thing we planned that we had to do.
+
−
 
+
−
                            We tried our best, even if it's hard to do we
+
−
 
+
−
                            never regret any decisions we have made
+
−
 
+
−
                            Cuz it worth it,it worth it, hey
+
−
 
+
−
                            We do the same, thing we planned that we had to do.
+
−
 
+
−
                            We tried our best, even if it's hard to do we
+
−
 
+
−
                            never regret any decisions we have made
+
−
 
+
−
                            Cuz it worth it,it worth it, hey
+
−
                        </section>
+
−
                     </section>
+
−
 
+
−
                    <p>In addition, to make the process of knowing synthetic biology intriguing, our team designs an
+
−
                        aeroplane chess map. The rules are similar to the original ones. We make some changes in the layout
+
−
                        of our map and the ‘ accident’ cards. Here attached our game.</p>
+
 
                 </section>
 
                 </section>
  
 
                 <section>
 
                 <section>
−
                     <h1 class="title">Rules:</h1>
+
                     <h1 class="title">BBa_K4002007</h1>
−
                    <p>DNA: (HEREDITY) The next round will have the same number of points as this round</p>
+
−
                    <p>RNA: (HEREDITY) Subtract 1 step per round</p>
+
−
                    <p>CRISPR-CAS9: Subtract the number of steps in the next round from the number of steps in the current
+
−
                        round. Take the absolute value if the number of steps in the next round is less.</p>
+
−
                    <p>Vaccines: Immunize all effects（e.g. ｜6-5｜=1 or ｜5-6｜=1）</p>
+
−
                    <p>Saccharomyces Cerevisiae: (Fermentation) Double the number of points in the next round</p>
+
−
                    <p>Cell Division: Stepping on a bond piece will have a split function. When stepped on by other bonds
+
−
                        won’t die, instead of going to the starting point. It can be split into 2 small bond, but both of
+
−
                        them can only be counted as one.</p>
+
−
                    <p>Pectase: (CATALYSIS) Roll the dice twice in the next round, help to get to terminus faster.</p>
+
−
                    <p>Pectin: (VISCIDITY) Get stuck in place one round</p>
+
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                    <p>Virus: Has the ability of attacking other players</p>
+
−
                    <ol>
+
−
                        <li><p>one player can be designated to take one step back each turn</p></li>
+
−
                        <li><p>overtake a designated player</p></li>
+
−
                    </ol>
+
−
                </section>
+
  
−
                <section>
+
                     <h2 class="title2">== Profile ==</h2>
−
                     <h1 class="title">Map:</h1>
+
                     <p>Name: <i>Cas9+gRNA+HR-L-endo-pgaA-HR-R</i></p>
−
                     <img class="m-b-12" src="https://static.igem.org/mediawiki/2021/4/49/T--Xiamen_City--img_communication_6.jpg" alt="" style="width: 50%;">
+
                    <p>Base Pairs: 7360 bp</p>
 +
                    <p>Origin: Synthetic</p>
 +
                    <p>Properties: CRISPR technology build a type of multi-functional yeast</p>
  
−
                     <p>To further enhance our influence, we planned to contribute and publish our research paper on public
+
                    <h2 class="title2">== Usage and Biology ==</h2>
−
                         education website <i>Questioz</i>, our abstract are followings:</p>
+
                     <p>Saccharomyces cerevisiae is a species of yeast (single-celled fungus microorganisms). The species has
−
                </section>
+
                        been instrumental in winemaking, baking, and brewing since ancient times. In fruit wine production,
 +
                         <i>Saccharomyces cerevisiae</i> uses the sugars in fruit juice to ferment to produce alcohol. It is
 +
                        necessary to add pectinase to destroy the pectin in the cell wall to increase the juice yield and
 +
                        increase the dissolution of aromatic substances such as pigments or terpenes.</p>
 +
                    <p>CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are specific regions in some
 +
                        bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes,
 +
                        function as an adaptive immune system in prokaryotes. While the specific ‘adaptive’ nature of this
 +
                        immunity is still under investigation, it is known that exogenous DNA is processed by Cas proteins
 +
                        into short (~30 base pair) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site.
 +
                        These short pieces of DNA are then incorporated into the host genome between repeat sequences to
 +
                        form spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs or
 +
                        pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then
 +
                        loaded into Cas proteins and act to guide them to initiate the sequence-specific cleavage of the
 +
                        target sequence.</p>
  
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                <section>
+
                     <div class="img-container m-t-12 m-b-12">
−
                     <h1 class="title">Background:</h1>
+
                        <img src="https://static.igem.org/mediawiki/2021/3/37/T--Xiamen_City--img_contribution_4.jpg" alt="" style="width: 80%;">
−
                    <p>During pandemic, transportation and trading has been greatly blocked . As commodity are deteriorating
+
                         <span class="figure">Figure 1 The principle of CRISPR Cas9.</span>
−
                        rapidly, fruit market has been greatly impacted. Many fruits were heavily stagnated and fruit
+
                    </div>
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                        farmers lost stable income. This study produces a new saccharomyces cerevisiae that might offers a
+
−
                         solution to this problem.</p>
+
−
                </section>
+
  
−
                <section>
+
                     <h2 class="title2">== Construct design ==</h2>
−
                     <h1 class="title">Methods: </h1>
+
                     <p>pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. sgRNA is a sequence of
−
                     <p>After two rounds of previous research, interviewing several bioengineering and zymurgy experts, we
+
                         RNA participant in CRISPR. HR-L is the homology arm upstream HXK1. HR-R is the homology arm
−
                         found this problem can be solved indirectly in biological method. Researchers are looking forward to
+
                         downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 2). </p>
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                        genetically-edit saccharomyces cerevisiae so it acquires pectinase’s function. This new yeast is
+
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                        expected to cut fruit wine production cost in order to lower wine industry’s barrier and finally
+
−
                        increase fruit demand. </p>
+
−
                    <p>Using CRISPR-Cas9 technology, the heterologous gene expression process of endo-PGAA gene from
+
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                        Aspergillus Niger SC323 in wine yeast was introduced into phcas9-Nours plasmid containing Cas gene.
+
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                        Then, the pYES2 -- GRNA-HYg-MCS plasmid carrying sgRNA (anchoring HXK1) and the homologous arm for
+
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                         repair were introduced into the fruit wine yeast. Lastly, the transposers were obtained to verify
+
−
                        whether endo-PGAA was replaced to the HXK1 gene site.</p>
+
−
                    <p>The process of verifying the ability of saccharomyces cerevisiae to produce pectinase are followings.
+
−
                        Bromophenol blue medium is used to verify whether the integrated strain produced pectinase. The
+
−
                        integrated strains are inoculated to 30°C incubator and cultured for 3 days. If we found that the
+
−
                        integrated strains on bromophenol blue medium had obvious yellow circles, while the wild type
+
−
                        strains did not, we can indicate that endo-PGAA gene has been successfully introduced into
+
−
                        saccharomyces cerevisiae.</p>
+
−
                </section>
+
−
 
+
−
                <section>
+
−
                    <h1 class="title">Results:</h1>
+
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                    <p>The experiment turned out to be successful. The final product can degrade pectin well and replace
+
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                        pectin in wine production process. Researchers are looking forward to commercializing it in the near
+
−
                        future.</p>
+
−
                </section>
+
−
 
+
−
                <section>
+
−
                    <h1 class="title">Here attached some question samples we used while we are interviewing with professors
+
−
                        and passing-by.</h1>
+
−
                    <ol>
+
  
−
                        <li><p>What do you think of the recent wine market? What about fruit wines?</p></li>
+
                    <div class="img-container m-t-12 m-b-12">
−
                         <li><p>Is there any national policy (regulation) on the wine market now? (Recently)</p></li>
+
                         <img src="https://static.igem.org/mediawiki/2021/1/1b/T--Xiamen_City--img_contribution_5.jpg" alt="" style="width: 80%;">
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                        <li><p>Do you think wineries will buy our yeast?</p></li>
+
                         <span class="figure">Figure 2. Cas9+gRNA+HR-L-endo-pgaA-HR-R box.</span>
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                        <li><p>How to deal with expired fruit wines?</p></li>
+
                     </div>
−
                        <li><p>Will it affect the stagnant fruits?</p></li>
+
−
                         <li><p>Is it costly to make fruit wine now?</p></li>
+
−
                        <li><p>What do you think about gene editing technology (are you willing to consume genetically
+
−
                            edited food?)</p></li>
+
−
                        <li><p>Do you know the difference between fruit wine and other wines?</p></li>
+
−
                        <li><p>How often do you usually drink wine?</p></li>
+
−
                        <li><p>What do you think is the impact of using gene editing technology to reduce the price of fruit
+
−
                            wines on the sales of fruit wines: good/bad/not much?</p></li>
+
−
                     </ol>
+
 
                 </section>
 
                 </section>
 
             </section>
 
             </section>

Latest revision as of 06:42, 19 October 2021

Contribution

BBa_K4002002

== Profile BBa_K4002002==

Name: HXK1

Base Pairs: 1458 bp

Origin: Saccharomyces cerevisiae, genome

Properties: An enzyme catalyze the phosphorylation of hexose

== Usage and Biology ==

This is a sequence coding enzyme HXK1. This enzyme can catalyze the phosphorylation of hexose to hexose 6-phosphate (D-glucose 6-phosphate and D-fructose 6-phosphate, respectively). Besides, it can mediate the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose 6-phosphate.

BBa_K4002003

== Profile ==

Name: pYES2-gRNA-hyg-MCS

Base Pairs: 388 bp

Origin: From article, Addgene

Properties: A piece of RNA complementary to the target gene.

== Usage and Biology ==

BBa_K4002003 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with. And this sequence is inserted into plasmid vector.

== Construct design ==

sgRNA is inserted into plasmid vector. Plasmid sequence map is shown in Figure 1.

Figure 1. Schematic map of pYES2-gRNA-hyg-MCS plasmid.

BBa_K4002004

== Profile ==

Name: pHCas9-Nours

Base Pairs: 4837bp

Origin: Streptococcus pyogenes, Addgene

Properties: An endonuclease enzyme associated with the CRISPR.

== Usage and Biology ==

This is a sequence coding pHCas9 protein. This protein is a dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system. The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. The genes that encode the Cas9 protein and sgRNA were introduced into a cell and programmed to change its target gene. sgRNA has regions that are complementary to the target sequence. A complex consisting of sgRNA and Cas9 protein is formed inside the cell and binds to target sites.

BBa_K4002005

== Profile ==

Name: endo-pgaA

Base Pairs: 1113bp

Origin: Aspergillus niger SC323, genome

Properties: An enzyme degradation of pectin

==== Usage and Biology ====

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase. This part as a repair template DNA was connected with homology arm of HXK1.

BBa_K4002006

== Profile ==

Name: HR-L-endo-pgaA-HR-R

Base Pairs: 2135 bp

Origin: Ssynthetic

Properties: CRISPR technology repair template for build a type of multi-functional yeast

== Usage and Biology ==

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase. Pectin is a significant carbohydrate component that comprises plant cell walls. The brewer's yeast uses sugar in the fruit juice to produce alcohol, and pectinase destroys the pectin located in the cell wall to improve the juice yield and eliminate the cloudiness of the fruit wine. HR-L and HR-R are homology arm, refers to the flanking sequence on both sides of the HXK1.

== Construct design ==

HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 1 and 2).

Figure 1. HR-L-endo-pgaA-HR-R box.
Figure 2. Schematic map of HR-L-endo-pgaA-HR-R.

BBa_K4002007

== Profile ==

Name: Cas9+gRNA+HR-L-endo-pgaA-HR-R

Base Pairs: 7360 bp

Origin: Synthetic

Properties: CRISPR technology build a type of multi-functional yeast

== Usage and Biology ==

Saccharomyces cerevisiae is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. In fruit wine production, Saccharomyces cerevisiae uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are specific regions in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes, function as an adaptive immune system in prokaryotes. While the specific ‘adaptive’ nature of this immunity is still under investigation, it is known that exogenous DNA is processed by Cas proteins into short (~30 base pair) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to form spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs or pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and act to guide them to initiate the sequence-specific cleavage of the target sequence.

Figure 1 The principle of CRISPR Cas9.

== Construct design ==

pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. sgRNA is a sequence of RNA participant in CRISPR. HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 2).

Figure 2. Cas9+gRNA+HR-L-endo-pgaA-HR-R box.

Contact Info

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