Difference between revisions of "Team:CPU CHINA/Design"

Line 1: Line 1:
−
<html lang="en">
+
<!doctype html>
 +
<html>
  
 
<head>
 
<head>
−
     <meta charset="UTF-8">
+
     <meta charset='UTF-8'>
−
    <meta http-equiv="X-UA-Compatible" content="IE=edge">
+
     <meta name='viewport' content='width=device-width initial-scale=1'>
−
     <meta name="viewport" content="width=device-width, initial-scale=1.0">
+
 
−
     <link rel="stylesheet"
+
     <link href='https://fonts.loli.net/css?family=Open+Sans:400italic,700italic,700,400&subset=latin,latin-ext'
−
        href="https://2017.igem.org/wiki/index.php?title= Template:SCU_China/default_CSS &action=raw&ctype=text/css">
+
        rel='stylesheet' type='text/css' />
−
    <link rel="stylesheet"
+
     <style type='text/css'>
−
        href="https://2021.igem.org/wiki/index.php?title= Template:CPU_CHINA/reset_CSS &action=raw&ctype=text/css">
+
         html {
−
    <link rel="stylesheet"
+
             overflow-x: initial !important;
−
        href="https://2021.igem.org/wiki/index.php?title= Template:CPU_CHINA/common_CSS &action=raw&ctype=text/css">
+
−
     <link rel="stylesheet"
+
−
        href="https://2021.igem.org/wiki/index.php?title= Template:CPU_CHINA/contentCommon_CSS &action=raw&ctype=text/css">
+
−
    <style>
+
−
         #header #title {
+
−
             left: 35%;
+
 
         }
 
         }
  
−
         #contain .left img {
+
         :root {
 +
            --bg-color: #ffffff;
 +
            --text-color: #333333;
 +
            --select-text-bg-color: #B5D6FC;
 +
            --select-text-font-color: auto;
 +
            --monospace: "Lucida Console", Consolas, "Courier", monospace;
 +
            --title-bar-height: 20px;
 +
        }
 +
 
 +
        .mac-os-11 {
 +
            --title-bar-height: 28px;
 +
        }
 +
 
 +
        html {
 +
            font-size: 14px;
 +
            background-color: var(--bg-color);
 +
            color: var(--text-color);
 +
            font-family: "Helvetica Neue", Helvetica, Arial, sans-serif;
 +
            -webkit-font-smoothing: antialiased;
 +
        }
 +
 
 +
        body {
 +
            margin: 0px;
 +
            padding: 0px;
 +
            height: auto;
 +
            inset: 0px;
 +
            font-size: 1rem;
 +
            line-height: 1.42857;
 +
            overflow-x: hidden;
 +
            background: inherit;
 +
            tab-size: 4;
 +
        }
 +
 
 +
        iframe {
 +
            margin: auto;
 +
        }
 +
 
 +
        a.url {
 +
            word-break: break-all;
 +
        }
 +
 
 +
        a:active,
 +
        a:hover {
 +
            outline: 0px;
 +
        }
 +
 
 +
        .in-text-selection,
 +
        ::selection {
 +
            text-shadow: none;
 +
            background: var(--select-text-bg-color);
 +
            color: var(--select-text-font-color);
 +
        }
 +
 
 +
        #write {
 +
            margin: 0px auto;
 +
            height: auto;
 +
            width: inherit;
 +
            word-break: normal;
 +
            overflow-wrap: break-word;
 +
            position: relative;
 +
            white-space: normal;
 +
            overflow-x: visible;
 +
            padding-top: 36px;
 +
        }
 +
 
 +
        #write.first-line-indent p {
 +
            text-indent: 2em;
 +
        }
 +
 
 +
        #write.first-line-indent li p,
 +
        #write.first-line-indent p * {
 +
            text-indent: 0px;
 +
        }
 +
 
 +
        #write.first-line-indent li {
 +
            margin-left: 2em;
 +
        }
 +
 
 +
        .for-image #write {
 +
            padding-left: 8px;
 +
            padding-right: 8px;
 +
        }
 +
 
 +
        body.typora-export {
 +
            padding-left: 30px;
 +
            padding-right: 30px;
 +
        }
 +
 
 +
        .typora-export .footnote-line,
 +
        .typora-export li,
 +
        .typora-export p {
 +
            white-space: pre-wrap;
 +
        }
 +
 
 +
        .typora-export .task-list-item input {
 +
            pointer-events: none;
 +
        }
 +
 
 +
        @media screen and (max-width: 500px) {
 +
            body.typora-export {
 +
                padding-left: 0px;
 +
                padding-right: 0px;
 +
            }
 +
 
 +
            #write {
 +
                padding-left: 20px;
 +
                padding-right: 20px;
 +
            }
 +
 
 +
            .CodeMirror-sizer {
 +
                margin-left: 0px !important;
 +
            }
 +
 
 +
            .CodeMirror-gutters {
 +
                display: none !important;
 +
            }
 +
        }
 +
 
 +
        #write li>figure:last-child {
 +
            margin-bottom: 0.5rem;
 +
        }
 +
 
 +
        #write ol,
 +
        #write ul {
 +
            position: relative;
 +
        }
 +
 
 +
        img {
 +
            max-width: 100%;
 +
            vertical-align: middle;
 +
            image-orientation: from-image;
 +
        }
 +
 
 +
        button,
 +
        input,
 +
        select,
 +
        textarea {
 +
            color: inherit;
 +
            font: inherit;
 +
        }
 +
 
 +
        input[type="checkbox"],
 +
        input[type="radio"] {
 +
            line-height: normal;
 +
            padding: 0px;
 +
        }
 +
 
 +
        *,
 +
        ::after,
 +
        ::before {
 +
            box-sizing: border-box;
 +
        }
 +
 
 +
        #write h1,
 +
        #write h2,
 +
        #write h3,
 +
        #write h4,
 +
        #write h5,
 +
        #write h6,
 +
        #write p,
 +
        #write pre {
 +
            width: inherit;
 +
        }
 +
 
 +
        #write h1,
 +
        #write h2,
 +
        #write h3,
 +
        #write h4,
 +
        #write h5,
 +
        #write h6,
 +
        #write p {
 +
            position: relative;
 +
        }
 +
 
 +
        p {
 +
            line-height: inherit;
 +
        }
 +
 
 +
        h1,
 +
        h2,
 +
        h3,
 +
        h4,
 +
        h5,
 +
        h6 {
 +
            break-after: avoid-page;
 +
            break-inside: avoid;
 +
            orphans: 4;
 +
        }
 +
 
 +
        p {
 +
            orphans: 4;
 +
        }
 +
 
 +
        h1 {
 +
            font-size: 2rem;
 +
        }
 +
 
 +
        h2 {
 +
            font-size: 1.8rem;
 +
        }
 +
 
 +
        h3 {
 +
            font-size: 1.6rem;
 +
        }
 +
 
 +
        h4 {
 +
            font-size: 1.4rem;
 +
        }
 +
 
 +
        h5 {
 +
            font-size: 1.2rem;
 +
        }
 +
 
 +
        h6 {
 +
            font-size: 1rem;
 +
        }
 +
 
 +
        .md-math-block,
 +
        .md-rawblock,
 +
        h1,
 +
        h2,
 +
        h3,
 +
        h4,
 +
        h5,
 +
        h6,
 +
        p {
 +
            margin-top: 1rem;
 +
            margin-bottom: 1rem;
 +
        }
 +
 
 +
        .hidden {
 +
            display: none;
 +
        }
 +
 
 +
        .md-blockmeta {
 +
            color: rgb(204, 204, 204);
 +
            font-weight: 700;
 +
            font-style: italic;
 +
        }
 +
 
 +
        a {
 +
            cursor: pointer;
 +
        }
 +
 
 +
        sup.md-footnote {
 +
            padding: 2px 4px;
 +
            background-color: rgba(238, 238, 238, 0.7);
 +
            color: rgb(85, 85, 85);
 +
            border-radius: 4px;
 +
            cursor: pointer;
 +
        }
 +
 
 +
        sup.md-footnote a,
 +
        sup.md-footnote a:hover {
 +
            color: inherit;
 +
            text-transform: inherit;
 +
            text-decoration: inherit;
 +
        }
 +
 
 +
        #write input[type="checkbox"] {
 +
            cursor: pointer;
 +
            width: inherit;
 +
            height: inherit;
 +
        }
 +
 
 +
        figure {
 +
            overflow-x: auto;
 +
            margin: 1.2em 0px;
 +
            max-width: calc(100% + 16px);
 +
            padding: 0px;
 +
        }
 +
 
 +
        figure>table {
 +
            margin: 0px;
 +
        }
 +
 
 +
        tr {
 +
            break-inside: avoid;
 +
            break-after: auto;
 +
        }
 +
 
 +
        thead {
 +
            display: table-header-group;
 +
        }
 +
 
 +
        table {
 +
            border-collapse: collapse;
 +
            border-spacing: 0px;
 +
            width: 100%;
 +
            overflow: auto;
 +
            break-inside: auto;
 +
            text-align: left;
 +
        }
 +
 
 +
        table.md-table td {
 +
            min-width: 32px;
 +
        }
 +
 
 +
        .CodeMirror-gutters {
 +
            border-right: 0px;
 +
            background-color: inherit;
 +
        }
 +
 
 +
        .CodeMirror-linenumber {
 +
            user-select: none;
 +
        }
 +
 
 +
        .CodeMirror {
 +
            text-align: left;
 +
        }
 +
 
 +
        .CodeMirror-placeholder {
 +
            opacity: 0.3;
 +
        }
 +
 
 +
        .CodeMirror pre {
 +
            padding: 0px 4px;
 +
        }
 +
 
 +
        .CodeMirror-lines {
 +
            padding: 0px;
 +
        }
 +
 
 +
        div.hr:focus {
 +
            cursor: none;
 +
        }
 +
 
 +
        #write pre {
 +
            white-space: pre-wrap;
 +
        }
 +
 
 +
        #write.fences-no-line-wrapping pre {
 +
            white-space: pre;
 +
        }
 +
 
 +
        #write pre.ty-contain-cm {
 +
            white-space: normal;
 +
        }
 +
 
 +
        .CodeMirror-gutters {
 +
            margin-right: 4px;
 +
        }
 +
 
 +
        .md-fences {
 +
            font-size: 0.9rem;
 +
            display: block;
 +
            break-inside: avoid;
 +
            text-align: left;
 +
            overflow: visible;
 +
            white-space: pre;
 +
            background: inherit;
 +
            position: relative !important;
 +
        }
 +
 
 +
        .md-fences-adv-panel {
 +
            width: 100%;
 +
            margin-top: 10px;
 +
            text-align: center;
 +
            padding-top: 0px;
 +
            padding-bottom: 8px;
 +
            overflow-x: auto;
 +
        }
 +
 
 +
        #write .md-fences.mock-cm {
 +
            white-space: pre-wrap;
 +
        }
 +
 
 +
        .md-fences.md-fences-with-lineno {
 +
            padding-left: 0px;
 +
        }
 +
 
 +
        #write.fences-no-line-wrapping .md-fences.mock-cm {
 +
            white-space: pre;
 +
            overflow-x: auto;
 +
        }
 +
 
 +
        .md-fences.mock-cm.md-fences-with-lineno {
 +
            padding-left: 8px;
 +
        }
 +
 
 +
        .CodeMirror-line,
 +
        twitterwidget {
 +
            break-inside: avoid;
 +
        }
 +
 
 +
        .footnotes {
 +
            opacity: 0.8;
 +
            font-size: 0.9rem;
 +
            margin-top: 1em;
 +
            margin-bottom: 1em;
 +
        }
 +
 
 +
        .footnotes+.footnotes {
 +
            margin-top: 0px;
 +
        }
 +
 
 +
        .md-reset {
 +
            margin: 0px;
 +
            padding: 0px;
 +
            border: 0px;
 +
            outline: 0px;
 +
            vertical-align: top;
 +
            background: 0px 0px;
 +
            text-decoration: none;
 +
            text-shadow: none;
 +
            float: none;
 +
            position: static;
 +
            width: auto;
 +
            height: auto;
 +
            white-space: nowrap;
 +
            cursor: inherit;
 +
            -webkit-tap-highlight-color: transparent;
 +
            line-height: normal;
 +
            font-weight: 400;
 +
            text-align: left;
 +
            box-sizing: content-box;
 +
            direction: ltr;
 +
        }
 +
 
 +
        li div {
 +
            padding-top: 0px;
 +
        }
 +
 
 +
        blockquote {
 +
            margin: 1rem 0px;
 +
        }
 +
 
 +
        li .mathjax-block,
 +
        li p {
 +
            margin: 0.5rem 0px;
 +
        }
 +
 
 +
        li blockquote {
 +
            margin: 1rem 0px;
 +
        }
 +
 
 +
        li {
 +
            margin: 0px;
 +
            position: relative;
 +
        }
 +
 
 +
        blockquote> :last-child {
 +
            margin-bottom: 0px;
 +
        }
 +
 
 +
        blockquote> :first-child,
 +
        li> :first-child {
 +
            margin-top: 0px;
 +
        }
 +
 
 +
        .footnotes-area {
 +
            color: rgb(136, 136, 136);
 +
            margin-top: 0.714rem;
 +
            padding-bottom: 0.143rem;
 +
            white-space: normal;
 +
        }
 +
 
 +
        #write .footnote-line {
 +
            white-space: pre-wrap;
 +
        }
 +
 
 +
        @media print {
 +
 
 +
            body,
 +
            html {
 +
                border: 1px solid transparent;
 +
                height: 99%;
 +
                break-after: avoid;
 +
                break-before: avoid;
 +
                font-variant-ligatures: no-common-ligatures;
 +
            }
 +
 
 +
            #write {
 +
                margin-top: 0px;
 +
                padding-top: 0px;
 +
                border-color: transparent !important;
 +
            }
 +
 
 +
            .typora-export * {
 +
                -webkit-print-color-adjust: exact;
 +
            }
 +
 
 +
            .typora-export #write {
 +
                break-after: avoid;
 +
            }
 +
 
 +
            .typora-export #write::after {
 +
                height: 0px;
 +
            }
 +
 
 +
            .is-mac table {
 +
                break-inside: avoid;
 +
            }
 +
 
 +
            .typora-export-show-outline .typora-export-sidebar {
 +
                display: none;
 +
            }
 +
        }
 +
 
 +
        .footnote-line {
 +
            margin-top: 0.714em;
 +
            font-size: 0.7em;
 +
        }
 +
 
 +
        a img,
 +
        img a {
 +
            cursor: pointer;
 +
        }
 +
 
 +
        pre.md-meta-block {
 +
            font-size: 0.8rem;
 +
            min-height: 0.8rem;
 +
            white-space: pre-wrap;
 +
            background: rgb(204, 204, 204);
 +
            display: block;
 +
            overflow-x: hidden;
 +
        }
 +
 
 +
        p>.md-image:only-child:not(.md-img-error) img,
 +
        p>img:only-child {
 +
            display: block;
 +
            margin: auto;
 +
        }
 +
 
 +
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        <h1 id="title">DESIGN</h1>
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            <nav>
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                <div class="catalogue-title">DESIGN</div>
+
−
                <ul class="catalogue">
+
−
                    <li class="num">
+
−
                        <a href="#section1" title="OVERVIEW">OVERVIEW</a>
+
−
                    </li>
+
−
                    <li class="num">
+
−
                        <a href="#section2" title="TO DETERMINE A CENTRAL PE DEGRADATION ELEMENT">TO DETERMINE A CENTRAL
+
−
                            PE DEGRADATION ELEMENT</a>
+
−
                    </li>
+
−
                    <li class="num">
+
−
                        <a href="#section3" title="TO ENHANCE THE PE-DEGRADING EFFICIENCY OF MnP">TO ENHANCE THE
+
−
                            PE-DEGRADING EFFICIENCY OF MnP</a>
+
−
                    </li>
+
−
                    <li class="num">
+
−
                        <a href="#section4" title="CONVERGE THE ADVANTAGES OF THREE FUNCTIONAL PROTEINS">CONVERGE THE
+
−
                            ADVANTAGES OF THREE FUNCTIONAL PROTEINS</a>
+
−
                    </li>
+
−
                    <li class="num">
+
−
                        <a href="#section5" title="THE OVERALL DIAGRAM">THE OVERALL DIAGRAM</a>
+
−
                    </li>
+
−
                </ul>
+
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            </nav>
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            <img src="https://static.igem.org/mediawiki/2021/b/b2/T--CPU_CHINA--experimentalize.png" alt="">
+
−
        </div>
+
−
        <div id="detail" class="clearfix">
+
  
−
             <div class="section" id="section1">
+
        .md-toc {
−
                <h2 class="mume-header" id="overview">OVERVIEW</h2>
+
             min-height: 3.58rem;
 +
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 +
            font-size: 0.9rem;
 +
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−
                <p>Currently, the major disposal methods for PE are <strong>incineration</strong> and
+
        .md-toc-content {
−
                    <strong>landfill</strong>,
+
             position: relative;
−
                    both of which are not the optimal way of disposing PE, for these two methods have led to
+
             margin-left: 0px;
−
                    <strong>negative
+
        }
−
                        environmental consequences</strong> not limited to the <strong>release of hazardous
+
−
                        substances</strong>, and the
+
−
                    <strong>occupancy of enormous land resources</strong>.
+
−
                </p>
+
−
                <p>Therefore, we decided to take advantage of the power of nature, seeking <strong>specific
+
−
                        agents</strong> that
+
−
                    possess the unique <strong>ability of degrading PE</strong>, and further <strong>modify</strong> and
+
−
                    <strong>optimize</strong> it to realize green and efficient degradation of PE.
+
−
                </p>
+
−
             </div>
+
−
             <div class="section" id="section2">
+
−
                <h2 class="mume-header" id="to-determine-a-central-pe-degradation-element">TO DETERMINE A CENTRAL PE
+
−
                    DEGRADATION
+
−
                    ELEMENT</h2>
+
  
−
                <h3 class="mume-header" id="agent-selection">AGENT SELECTION</h3>
+
        .md-toc-content::after,
 +
        .md-toc::after {
 +
            display: none;
 +
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−
                <p>During our preliminary stage of literature research, strains of microorganism as well as enzymes that
+
        .md-toc-item {
−
                    both had
+
            display: block;
−
                    the <strong>potential of PE degradation</strong> were obtained by us. An either-or decision must be
+
            color: rgb(65, 131, 196);
−
                    made upon the
+
        }
−
                    selection of the PE-degrading agent. Without much hesitation, we selected <strong>enzymes</strong>
+
−
                    instead of
+
−
                    strains due to a more definite origin and characteristics provided by online databases. After
+
−
                    screening through
+
−
                    potential candidates, the very <strong>manganese peroxidase</strong> (MnP) was selected as our
+
−
                    <strong>central
+
−
                        functional element</strong>.</p>
+
−
                <p>It is a highly glycosylated lignin peroxidase with heme<sup>[1,2]</sup> that can <strong>oxidize
+
−
                        Mn<sup>2+</sup>
+
−
                        to Mn<sup>3+</sup></strong>, the latter can be <strong>chelated</strong> by ligands like
+
−
                    <strong>oxalic
+
−
                        acid</strong>, forming the <strong>Mn<sup>3+</sup>-ligand chelate compound</strong> that can
+
−
                    <strong>diffuse</strong> outside the enzyme for further degradation of lignin or other refractory
+
−
                    chemicals<sup>[3]</sup>.
+
−
                </p>
+
−
                <img src="https://static.igem.org/mediawiki/2021/5/5e/T--CPU_CHINA--K3853008_Fig1.png" alt="">
+
−
                <p class="imgdescribe"><strong>Fig. 1 The catalytic cycle of MnP.</strong></p>
+
−
                <p>It has been reported that <strong>MnP has a significant degradation efficiency on PE film</strong>.
+
−
                    As reported
+
−
                    before, the weight-average molecular weight (Mw) of PE was halved by MnP treatment for two days,
+
−
                    showing its
+
−
                    <strong>remarkable degradation efficacy</strong><sup>[4]</sup>. Thus, MnP was chosen by us as the
+
−
                    key element for
+
−
                    PE degradation.
+
−
                </p>
+
−
                <h3 class="mume-header" id="using-aao-as-a-better-approach-to-provide-substrate-for-mnp">USING AAO AS A
+
−
                    BETTER
+
−
                    APPROACH TO PROVIDE SUBSTRATE FOR MnP</h3>
+
  
−
                <p>It is shown on the catalytic cycle of MnP above that <strong>H<sub>2</sub>O<sub>2</sub></strong> is
+
        .md-toc-item a {
−
                    required as
+
            text-decoration: none;
−
                    its <strong>essential substrate</strong> for activating the enzymatic reaction. Yet an
+
        }
−
                    <strong>abnormally
+
−
                        high</strong> concentration of H<sub>2</sub>O<sub>2</sub> could also <strong>inhibit</strong>,
+
−
                    even
+
−
                    <strong>deactivate</strong> the enzyme, which might happen when H<sub>2</sub>O<sub>2</sub> was added
+
−
                    into the
+
−
                    system manually and periodically.
+
−
                </p>
+
−
                <p>Therefore, a more in-depth investigation was carried out to seek a solution. As a result, we
+
−
                    discovered a
+
−
                    specific type of enzyme, namely <strong>aryl alcohol oxidase (AAO)</strong>. It is an enzyme
+
−
                    containing
+
−
                    flavin-adenine-dinucleotide (FAD) that catalyzes the oxidation of aromatic and aliphatic allylic
+
−
                    primary alcohols
+
−
                    (which are far less oxidative when compared to Mn<sup>3+</sup> and H<sub>2</sub>O<sub>2</sub>) to
+
−
                    the
+
−
                    corresponding aldehydes while <strong>reducing molecular oxygen to
+
−
                        H<sub>2</sub>O<sub>2</sub></strong>.</p>
+
−
                <img src="https://static.igem.org/mediawiki/2021/e/e7/T--CPU_CHINA--BBa_K3853009_fig_1.png" alt="">
+
−
                <p class="imgdescribe"><strong>Fig. 2 The mechanism of AAO reducing molecular oxygen to
+
−
                        H<sub>2</sub>O<sub>2</sub> by oxidizing
+
−
                        4-methoxybenzyl alcohol.</strong></p>
+
−
                <p>We learned from the literature that AAO is able to produce H<sub>2</sub>O<sub>2</sub> in a
+
−
                    <strong>low but
+
−
                        steady</strong> rate. Therefore, the <strong>inhibition</strong> of MnP <strong>due to</strong>
+
−
                    <strong>an</strong> <strong>excess of H<sub>2</sub>O<sub>2</sub></strong> concentration can be
+
−
                    <strong>effectively
+
−
                        prevented</strong> when applying AAO as the source of H<sub>2</sub>O<sub>2</sub>. This would
+
−
                    allow MnP to
+
−
                    <strong>catalyze</strong> the PE-degrading reaction <strong>over a longer period of time</strong>,
+
−
                    realizing a
+
−
                    more <strong>complete degradation</strong> of PE. In addition, since the two enzymes work in tandem,
+
−
                    the
+
−
                    <strong>cascade reaction</strong> mediated by the two can only be initiated when substrates of AAO
+
−
                    is introduced
+
−
                    to the system. Therefore, we can <strong>achieve precise control</strong> to the onset and
+
−
                    termination of the
+
−
                    reactions via adding specific amount of substrates to the system in a given time, preventing
+
−
                    uncontrollable
+
−
                    situations from happening. As a result, we decided to select AAO as the assistant of MnP.
+
−
                </p>
+
−
                <img src="https://static.igem.org/mediawiki/2021/a/a4/T--CPU_CHINA--Design-3.jpg" alt="">
+
−
                <p class="imgdescribe"><strong>Fig. 3 The synergistic PE degradation effect of MnP and AAO.</strong></p>
+
−
            </div>
+
−
            <div class="section" id="section3">
+
−
                <h2 class="mume-header" id="to-enhance-the-pe-degrading-efficiency-of-mnp">TO ENHANCE THE PE-DEGRADING
+
−
                    EFFICIENCY OF
+
−
                    MnP</h2>
+
  
−
                <h3 class="mume-header" id="optimize-the-degradation-competence-of-mnp-by-directed-evolution">OPTIMIZE
+
        .md-toc-inner:hover {
−
                    THE
+
            text-decoration: underline;
−
                    DEGRADATION COMPETENCE OF MnP BY DIRECTED EVOLUTION</h3>
+
        }
  
−
                <p>As our key PE-degrading enzyme, manganese peroxidase (MnP) undertakes a <strong>fundamental</strong>
+
        .md-toc-inner {
−
                    role of
+
            display: inline-block;
−
                    inflicting oxidation to PE by continuously producing Mn<sup>3+</sup> ions. Therefore,
+
            cursor: pointer;
−
                    <strong>enhancing</strong>
+
        }
−
                    the degradation <strong>efficiency</strong> of MnP is beneficial to reach <strong>a more complete
+
−
                        destruction</strong> of PE films.</p>
+
−
                <p>In theory, there are <strong>two approaches</strong> of reinforce the degradation efficacy of MnP,
+
−
                    whether by
+
−
                    <strong>increasing the activity</strong> of MnP to realize a stronger oxidative capacity, or by
+
−
                    <strong>improving
+
−
                        the stability</strong> of MnP to prolong its duration of effect. However, since the substrate
+
−
                    and catalysate of
+
−
                    MnP are both highly-oxidative, simply increasing its activity without restrictions is bound to cause
+
−
                    irreversible
+
−
                    harm not only to the MnP itself, but also to other affiliated elements in our design, AAO for
+
−
                    instance. Therefore,
+
−
                    we decided to <strong>improve the stability</strong> of MnP by proposing a <strong>semi-rational
+
−
                        directed
+
−
                        evolution strategy</strong> towards it, with the hope to increase its tolerance of high
+
−
                    temperature, acidic pH,
+
−
                    as well as different types of organic solvents, all of which are common inhibitory physiochemical
+
−
                    properties that
+
−
                    may severely impact the activity of MnP.
+
−
                </p>
+
−
                <p>For the results of our directed-evolution attempt, see <strong><em><a href="https://2021.igem.org/Team:CPU_CHINA/Improve">Improvement</a></em>
+
−
                        </strong>
+
−
                    page</p>
+
−
                <h3 class="mume-header" id="facilitate-the-surface-adherence-of-mnp-by-introducing-hfb1">FACILITATE THE
+
−
                    SURFACE
+
−
                    ADHERENCE OF MnP BY INTRODUCING HFB1</h3>
+
  
−
                <p>Back to the stage where we were searching for agents with PE degradation efficacy, we noticed that
+
        .md-toc-h1 .md-toc-inner {
−
                    certain
+
            margin-left: 0px;
−
                    bacterial or fungal strains capable of degrading PE could produce <strong>biosurfactant</strong> to
+
             font-weight: 700;
−
                    assist their
+
        }
−
                    adherence and colonization on the hydrophobic surface of plastics, so that they could degrade PE in
+
−
                    a faster pace.
+
−
                    This inspired us to introduce biosurfactant into our design, aiming to <strong>increase the
+
−
                        hydrophilicity</strong> of the surface of PE.</p>
+
−
                <p>As a result, our focus was concentrated on hydrophobin-1 (HFB1), a kind of class &#x2161; HFBs
+
−
                    derived from
+
−
                    <em>Trichoderma reesei</em>. It is rich in hydrophobic amino acids, endowing its surface activity.
+
−
                    By
+
−
                    <strong>self-assembling</strong> at hydrophilic-hydrophobic interfaces autonomously, HFB1 can
+
−
                    <strong>enhance the
+
−
                        affinity</strong> between hydrophilic proteins and hydrophobic materials <strong>such as
+
−
                        PE</strong>, thus
+
−
                    facilitating its contact with aqueous environment, thereby facilitating MnP to degrade PE.
+
−
                </p>
+
−
                <p>What&apos;s more, compared with other members of HFBs, HFB1 has <strong>better stability</strong> and
+
−
                    <strong>higher surface activity</strong>, which means it can maintain its function of adherence on
+
−
                    hydrophobic
+
−
                    substances <strong>more firmly</strong> for a <strong>longer period of time</strong>.
+
−
                </p>
+
−
                <p>Therefore, in our project, HFB1 is selected and used as a biosurfactant to produce consistent surface
+
−
                    activity on
+
−
                    PE, thereby promoting the adherence of MnP on PE surface, which helps to <strong>improve</strong>
+
−
                    the degradation
+
−
                    <strong>efficacy</strong> of this enzyme.
+
−
                </p>
+
−
             </div>
+
−
            <div class="section" id="section4">
+
−
                <h2 class="mume-header" id="converge-the-advantages-of-three-functional-proteins">CONVERGE THE
+
−
                    ADVANTAGES OF THREE
+
−
                    FUNCTIONAL PROTEINS</h2>
+
  
−
                <p>Now that the three functional proteins were selected, all of which possesses individual functions
+
        .md-toc-h2 .md-toc-inner {
−
                    that could
+
            margin-left: 2em;
−
                    contribute to the degradation of PE, instead of directly applying all of them by simply adding them
+
        }
−
                    into the
+
−
                    system separately, we began to consider the possibility of <strong>combining</strong> these discrete
+
−
                    parts into a
+
−
                    <strong>composite entity</strong>, enabling the production of a strong synergistic effect which may
+
−
                    lead to an
+
−
                    significant improvement on efficacy.
+
−
                </p>
+
−
                <h3 class="mume-header" id="getting-closer-to-the-surface-of-pe">GETTING CLOSER TO THE SURFACE OF PE
+
−
                </h3>
+
  
−
                <p>The first idea that struck us was that we could minimize the spatial distance between MnP and PE by
+
        .md-toc-h3 .md-toc-inner {
−
                    <strong>fusing HFB1</strong> on the enzyme. Similar strategy could also be applied on AAO to
+
            margin-left: 4em;
−
                    generate fusion
+
        }
−
                    protein as well. In this way, our functional enzymes can simultaneously be anchored to the PE
+
−
                    surface with the aid
+
−
                    of fused HFB1, so that the <strong>diffusion distance</strong> of Mn<sup>3+</sup>-ligand chelate
+
−
                    compound towards
+
−
                    PE could be <strong>significantly lessened</strong>, enabling a more efficient degradation outcome.
+
−
                    Meanwhile, the
+
−
                    H<sub>2</sub>O<sub>2</sub> generated by AAO can also become more accessible to MnP when the two
+
−
                    enzymes are
+
−
                    <strong>closely anchored</strong> to the surface of PE.
+
−
                </p>
+
−
                <p>Therefore, we delved into literatures and previous iGEM projects to look for ideal solutions. It
+
−
                    turned out that
+
−
                    there existed a versatile protein ligation system, i.e. <strong>SpyCatcher/SpyTag system</strong>,
+
−
                    that has been
+
−
                    widely adopted by many laboratories and iGEM teams for construction of multi-domain protein. This
+
−
                    system contains
+
−
                    <strong>two essential elements</strong>:
+
−
                </p>
+
−
                <ul>
+
−
                    <li><strong>SpyCatcher:</strong> a modified immunoglobulin-like domain CnaB2 from a
+
−
                        <em>Streptococcus
+
−
                            pyogenes</em> surface protein</li>
+
−
                    <li><strong>SpyTag:</strong> a cognate 13-amino-acid peptide</li>
+
−
                </ul>
+
−
                <img src="https://static.igem.org/mediawiki/2021/b/b6/T--CPU_CHINA--Design-4.1.png" alt="">
+
−
                <p class="imgdescribe"><strong>Fig. 4 The isopepide-forming mechanism between the two Spy
+
−
                        domains.</strong> <em>Glu77 &amp; Lys31 are
+
−
                        the residues on SpyCatcher; Asp117 is the residue on SpyTag.</em></p>
+
−
                <p>The two domains can <strong>autonomously form a covalent isopeptide bond</strong> between each other,
+
−
                    thereby
+
−
                    linking the two portions together. By linking the Spy domains on the N-terminal or C-terminal of the
+
−
                    target
+
−
                    protein with <strong>elastin-like protein</strong> (ELP) or <strong>serine/glycine link</strong>
+
−
                    (Ser/Gly link),
+
−
                    its structure and function are generally <strong>unaffected</strong>, while the formation of
+
−
                    isopeptide bond
+
−
                    between SpyCatcher and SpyTag remains effective and efficient. By adopting this system, MnP and AAO
+
−
                    that was fused
+
−
                    with HFB1 are able to stick to surface of PE, realizing a better spatial concentration on it.</p>
+
−
                <h3 class="mume-header" id="getting-closer-with-each-other">GETTING CLOSER WITH EACH OTHER</h3>
+
  
−
                <p>Albeit introducing SpyCatcher/SpyTag connect system into our design could have a positive influence
+
        .md-toc-h4 .md-toc-inner {
−
                    on
+
            margin-left: 6em;
−
                    accelerating PE degradation, several shortcomings are not yet solved. For example, the adherence of
+
        }
−
                    MnP-HFB1 and
+
−
                    AAO-HFB1 fusion proteins on the surface of PE are likely to be unordered instead of evenly
+
−
                    distributed. Protein
+
−
                    clusters of the same type of fusion protein are likely to be formed on the PE surface, preventing
+
−
                    thorough
+
−
                    substance exchange between discrete protein molecules. Also, the maintenance of optimum functioning
+
−
                    ratio between
+
−
                    MnP and AAO cannot be guaranteed due to the arbitrary distribution on the PE surface. Both of the
+
−
                    two
+
−
                    uncontrollable conditions will reduce the efficacy of PE degradation.</p>
+
−
                <img src="https://static.igem.org/mediawiki/2021/7/7e/T--CPU_CHINA--Design-5.PNG" alt="">
+
−
                <p class="imgdescribe"><strong>Fig. 5 The potentially huge differences between ideality and
+
−
                        reality.</strong></p>
+
−
                <p>To enable the binding of MnP and AAO on the surface of PE in a more organized manner, we adopted a
+
−
                    recently
+
−
                    reported CRISPR/Cas-based DNA anchoring system to our design. This system utilizes an deactivated
+
−
                    CRISPR-associated protein 9 linked to a SpyCatcher domain (dCas9-SpyCatcher), which can not only
+
−
                    form a covalent
+
−
                    bond with proteins fused with SpyTag domain, but also recognize and bind to complementary DNA
+
−
                    sequences after
+
−
                    incorporating a single-guide RNA (sgRNA) without cleavage activity.<br>
+
−
                    Therefore, by specially designing a double-stranded DNA (dsDNA) with multiple sequence segments
+
−
                    complementary to
+
−
                    different sgRNAs, the dCas9-SpyCatcher incorporated with different types of sgRNAs and functional
+
−
                    proteins can be
+
−
                    anchored to the double-stranded DNA in a predetermined number and proportion.</p>
+
−
                <p>In our eventual design, the three functional proteins are all fused with SpyTag, covalently linked
+
−
                    with
+
−
                    dCas9-SpyCatcher, and anchored to the same dsDNA. In this way, the spatial distance between MnP and
+
−
                    AAO could be
+
−
                    remarkably reduced. The close proximity and determined proportion between the two enzymes can
+
−
                    greatly facilitate
+
−
                    substance exchange, thereby releasing a steady flow of PE-oxidizing agent when given sufficient
+
−
                    substrate.
+
−
                    Moreover, instead of pulling one individual enzyme once for all, HFB1, or HFB1s, can now paste the
+
−
                    whole
+
−
                    protein-nucleic-acid complex onto the surface of PE synergistically.</p>
+
−
            </div>
+
−
            <div class="section" id="section5">
+
−
                <h2 class="mume-header" id="the-overall-diagram">THE OVERALL DIAGRAM</h2>
+
  
−
                 <p>Eventually, by combining the three PE-degrading elements with the two assembly systems, we are able
+
        .md-toc-h5 .md-toc-inner {
−
                     to construct
+
            margin-left: 8em;
−
                    a new type of protein-nucleic-acid complex that possesses an enhanced ability for PE degradation.
+
        }
−
                    And we name it,
+
 
−
                    polyethylene degradist.</p>
+
        .md-toc-h6 .md-toc-inner {
−
                <p>A demonstrative graph is shown below.</p>
+
            margin-left: 10em;
−
                <img src="https://static.igem.org/mediawiki/2021/e/ee/T--CPU_CHINA--Design-6.jpg" alt="">
+
        }
−
                <p class="imgdescribe"><strong>Fig. 6 The final conceptual design overview of our PE-degrading
+
 
−
                        system.</strong></p>
+
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            .typora-export-sidebar {
 +
                display: none;
 +
            }
 +
        }
 +
 
 +
        .outline-content li,
 +
        .outline-content ul {
 +
            margin-left: 0px;
 +
            margin-right: 0px;
 +
            padding-left: 0px;
 +
            padding-right: 0px;
 +
            list-style: none;
 +
        }
 +
 
 +
        .outline-content ul {
 +
            margin-top: 0px;
 +
            margin-bottom: 0px;
 +
        }
 +
 
 +
        .outline-content strong {
 +
            font-weight: 400;
 +
        }
 +
 
 +
        .outline-expander {
 +
            width: 1rem;
 +
            height: 1.42857rem;
 +
            position: relative;
 +
            display: table-cell;
 +
            vertical-align: middle;
 +
            cursor: pointer;
 +
            padding-left: 4px;
 +
        }
 +
 
 +
        .outline-expander::before {
 +
            content: "";
 +
            position: relative;
 +
            font-family: Ionicons;
 +
            display: inline-block;
 +
            font-size: 8px;
 +
            vertical-align: middle;
 +
        }
 +
 
 +
        .outline-item {
 +
            padding-top: 3px;
 +
            padding-bottom: 3px;
 +
            cursor: pointer;
 +
        }
 +
 
 +
        .outline-expander:hover::before {
 +
            content: "";
 +
        }
 +
 
 +
        .outline-h1>.outline-item {
 +
            padding-left: 0px;
 +
        }
 +
 
 +
        .outline-h2>.outline-item {
 +
            padding-left: 1em;
 +
        }
 +
 
 +
        .outline-h3>.outline-item {
 +
            padding-left: 2em;
 +
        }
 +
 
 +
        .outline-h4>.outline-item {
 +
            padding-left: 3em;
 +
        }
 +
 
 +
        .outline-h5>.outline-item {
 +
            padding-left: 4em;
 +
        }
 +
 
 +
        .outline-h6>.outline-item {
 +
            padding-left: 5em;
 +
        }
 +
 
 +
        .outline-label {
 +
            cursor: pointer;
 +
            display: table-cell;
 +
            vertical-align: middle;
 +
            text-decoration: none;
 +
            color: inherit;
 +
        }
 +
 
 +
        .outline-label:hover {
 +
            text-decoration: underline;
 +
        }
 +
 
 +
        .outline-item:hover {
 +
            border-color: rgb(245, 245, 245);
 +
            background-color: var(--item-hover-bg-color);
 +
        }
 +
 
 +
        .outline-item:hover {
 +
            margin-left: -28px;
 +
            margin-right: -28px;
 +
            border-left: 28px solid transparent;
 +
            border-right: 28px solid transparent;
 +
        }
 +
 
 +
        .outline-item-single .outline-expander::before,
 +
        .outline-item-single .outline-expander:hover::before {
 +
            display: none;
 +
        }
 +
 
 +
        .outline-item-open>.outline-item>.outline-expander::before {
 +
            content: "";
 +
        }
 +
 
 +
        .outline-children {
 +
            display: none;
 +
        }
 +
 
 +
        .info-panel-tab-wrapper {
 +
            display: none;
 +
        }
 +
 
 +
        .outline-item-open>.outline-children {
 +
            display: block;
 +
        }
 +
 
 +
        .typora-export .outline-item {
 +
            padding-top: 1px;
 +
            padding-bottom: 1px;
 +
        }
 +
 
 +
        .typora-export .outline-item:hover {
 +
            margin-right: -8px;
 +
            border-right: 8px solid transparent;
 +
        }
 +
 
 +
        .typora-export .outline-expander::before {
 +
            content: "+";
 +
            font-family: inherit;
 +
            top: -1px;
 +
        }
 +
 
 +
        .typora-export .outline-expander:hover::before,
 +
        .typora-export .outline-item-open>.outline-item>.outline-expander::before {
 +
            content: "−";
 +
        }
 +
 
 +
        .typora-export-collapse-outline .outline-children {
 +
            display: none;
 +
        }
 +
 
 +
        .typora-export-collapse-outline .outline-item-open>.outline-children,
 +
        .typora-export-no-collapse-outline .outline-children {
 +
            display: block;
 +
        }
 +
 
 +
        .typora-export-no-collapse-outline .outline-expander::before {
 +
            content: "" !important;
 +
        }
 +
 
 +
        .typora-export-show-outline .outline-item-active>.outline-item .outline-label {
 +
            font-weight: 700;
 +
        }
 +
 
 +
        .md-inline-math-container mjx-container {
 +
            zoom: 0.95;
 +
        }
 +
 
 +
 
 +
        :root {
 +
            --side-bar-bg-color: #fafafa;
 +
            --control-text-color: #777;
 +
        }
 +
 
 +
        @include-when-export url(https://fonts.loli.net/css?family=Open+Sans:400italic,700italic,700,400&subset=latin,latin-ext);
 +
 
 +
        /* open-sans-regular - latin-ext_latin */
 +
        /* open-sans-italic - latin-ext_latin */
 +
        /* open-sans-700 - latin-ext_latin */
 +
        /* open-sans-700italic - latin-ext_latin */
 +
        html {
 +
            font-size: 16px;
 +
            -webkit-font-smoothing: antialiased;
 +
        }
 +
 
 +
        body {
 +
            font-family: "Open Sans", "Clear Sans", "Helvetica Neue", Helvetica, Arial, 'Segoe UI Emoji', sans-serif;
 +
            color: rgb(51, 51, 51);
 +
            line-height: 1.6;
 +
        }
 +
 
 +
        #write {
 +
            max-width: 860px;
 +
            margin: 0 auto;
 +
            padding: 30px;
 +
            padding-bottom: 100px;
 +
        }
 +
 
 +
        @media only screen and (min-width: 1400px) {
 +
            #write {
 +
                max-width: 1024px;
 +
            }
 +
        }
 +
 
 +
        @media only screen and (min-width: 1800px) {
 +
            #write {
 +
                max-width: 1200px;
 +
            }
 +
        }
 +
 
 +
        #write>ul:first-child,
 +
        #write>ol:first-child {
 +
            margin-top: 30px;
 +
        }
 +
 
 +
        a {
 +
            color: #4183C4;
 +
        }
 +
 
 +
        h1,
 +
        h2,
 +
        h3,
 +
        h4,
 +
        h5,
 +
        h6 {
 +
            position: relative;
 +
            margin-top: 1rem;
 +
            margin-bottom: 1rem;
 +
            font-weight: bold;
 +
            line-height: 1.4;
 +
            cursor: text;
 +
        }
 +
 
 +
        h1:hover a.anchor,
 +
        h2:hover a.anchor,
 +
        h3:hover a.anchor,
 +
        h4:hover a.anchor,
 +
        h5:hover a.anchor,
 +
        h6:hover a.anchor {
 +
            text-decoration: none;
 +
        }
 +
 
 +
        h1 tt,
 +
        h1 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h2 tt,
 +
        h2 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h3 tt,
 +
        h3 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h4 tt,
 +
        h4 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h5 tt,
 +
        h5 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h6 tt,
 +
        h6 code {
 +
            font-size: inherit;
 +
        }
 +
 
 +
        h1 {
 +
            font-size: 2.25em;
 +
            line-height: 1.2;
 +
            border-bottom: 1px solid #eee;
 +
        }
 +
 
 +
        h2 {
 +
            font-size: 1.75em;
 +
            line-height: 1.225;
 +
            border-bottom: 1px solid #eee;
 +
        }
 +
 
 +
        /*@media print {
 +
    .typora-export h1,
 +
    .typora-export h2 {
 +
        border-bottom: none;
 +
        padding-bottom: initial;
 +
    }
 +
 
 +
    .typora-export h1::after,
 +
    .typora-export h2::after {
 +
        content: "";
 +
        display: block;
 +
        height: 100px;
 +
        margin-top: -96px;
 +
        border-top: 1px solid #eee;
 +
    }
 +
}*/
 +
 
 +
        h3 {
 +
            font-size: 1.5em;
 +
            line-height: 1.43;
 +
        }
 +
 
 +
        h4 {
 +
            font-size: 1.25em;
 +
        }
 +
 
 +
        h5 {
 +
            font-size: 1em;
 +
        }
 +
 
 +
        h6 {
 +
            font-size: 1em;
 +
            color: #777;
 +
        }
 +
 
 +
        p,
 +
        blockquote,
 +
        ul,
 +
        ol,
 +
        dl,
 +
        table {
 +
            margin: 0.8em 0;
 +
        }
 +
 
 +
        li>ol,
 +
        li>ul {
 +
            margin: 0 0;
 +
        }
 +
 
 +
        hr {
 +
            height: 2px;
 +
            padding: 0;
 +
            margin: 16px 0;
 +
            background-color: #e7e7e7;
 +
            border: 0 none;
 +
            overflow: hidden;
 +
            box-sizing: content-box;
 +
        }
 +
 
 +
        li p.first {
 +
            display: inline-block;
 +
        }
 +
 
 +
        ul,
 +
        ol {
 +
            padding-left: 30px;
 +
        }
 +
 
 +
        ul:first-child,
 +
        ol:first-child {
 +
            margin-top: 0;
 +
        }
 +
 
 +
        ul:last-child,
 +
        ol:last-child {
 +
            margin-bottom: 0;
 +
        }
 +
 
 +
        blockquote {
 +
            border-left: 4px solid #dfe2e5;
 +
            padding: 0 15px;
 +
            color: #777777;
 +
        }
 +
 
 +
        blockquote blockquote {
 +
            padding-right: 0;
 +
        }
 +
 
 +
        table {
 +
            padding: 0;
 +
            word-break: initial;
 +
        }
 +
 
 +
        table tr {
 +
            border: 1px solid #dfe2e5;
 +
            margin: 0;
 +
            padding: 0;
 +
        }
 +
 
 +
        table tr:nth-child(2n),
 +
        thead {
 +
            background-color: #f8f8f8;
 +
        }
 +
 
 +
        table th {
 +
            font-weight: bold;
 +
            border: 1px solid #dfe2e5;
 +
            border-bottom: 0;
 +
            margin: 0;
 +
            padding: 6px 13px;
 +
        }
 +
 
 +
        table td {
 +
            border: 1px solid #dfe2e5;
 +
            margin: 0;
 +
            padding: 6px 13px;
 +
        }
 +
 
 +
        table th:first-child,
 +
        table td:first-child {
 +
            margin-top: 0;
 +
        }
 +
 
 +
        table th:last-child,
 +
        table td:last-child {
 +
            margin-bottom: 0;
 +
        }
 +
 
 +
        .CodeMirror-lines {
 +
            padding-left: 4px;
 +
        }
 +
 
 +
        .code-tooltip {
 +
            box-shadow: 0 1px 1px 0 rgba(0, 28, 36, .3);
 +
            border-top: 1px solid #eef2f2;
 +
        }
 +
 
 +
        .md-fences,
 +
        code,
 +
        tt {
 +
            border: 1px solid #e7eaed;
 +
            background-color: #f8f8f8;
 +
            border-radius: 3px;
 +
            padding: 0;
 +
            padding: 2px 4px 0px 4px;
 +
            font-size: 0.9em;
 +
        }
 +
 
 +
        code {
 +
            background-color: #f3f4f4;
 +
            padding: 0 2px 0 2px;
 +
        }
 +
 
 +
        .md-fences {
 +
            margin-bottom: 15px;
 +
            margin-top: 15px;
 +
            padding-top: 8px;
 +
            padding-bottom: 6px;
 +
        }
 +
 
 +
 
 +
        .md-task-list-item>input {
 +
            margin-left: -1.3em;
 +
        }
 +
 
 +
        @media print {
 +
            html {
 +
                font-size: 13px;
 +
            }
 +
 
 +
            table,
 +
            pre {
 +
                page-break-inside: avoid;
 +
            }
 +
 
 +
            pre {
 +
                word-wrap: break-word;
 +
            }
 +
        }
 +
 
 +
        .md-fences {
 +
            background-color: #f8f8f8;
 +
        }
 +
 
 +
        #write pre.md-meta-block {
 +
            padding: 1rem;
 +
            font-size: 85%;
 +
            line-height: 1.45;
 +
            background-color: #f7f7f7;
 +
            border: 0;
 +
            border-radius: 3px;
 +
            color: #777777;
 +
            margin-top: 0 !important;
 +
        }
 +
 
 +
        .mathjax-block>.code-tooltip {
 +
            bottom: .375rem;
 +
        }
 +
 
 +
        .md-mathjax-midline {
 +
            background: #fafafa;
 +
        }
 +
 
 +
        #write>h3.md-focus:before {
 +
            left: -1.5625rem;
 +
            top: .375rem;
 +
        }
 +
 
 +
        #write>h4.md-focus:before {
 +
            left: -1.5625rem;
 +
            top: .285714286rem;
 +
        }
 +
 
 +
        #write>h5.md-focus:before {
 +
            left: -1.5625rem;
 +
            top: .285714286rem;
 +
        }
 +
 
 +
        #write>h6.md-focus:before {
 +
            left: -1.5625rem;
 +
            top: .285714286rem;
 +
        }
 +
 
 +
        .md-image>.md-meta {
 +
            /*border: 1px solid #ddd;*/
 +
            border-radius: 3px;
 +
            padding: 2px 0px 0px 4px;
 +
            font-size: 0.9em;
 +
            color: inherit;
 +
        }
 +
 
 +
        .md-tag {
 +
            color: #a7a7a7;
 +
            opacity: 1;
 +
        }
 +
 
 +
        .md-toc {
 +
            margin-top: 20px;
 +
            padding-bottom: 20px;
 +
        }
 +
 
 +
        .sidebar-tabs {
 +
            border-bottom: none;
 +
        }
 +
 
 +
        #typora-quick-open {
 +
            border: 1px solid #ddd;
 +
            background-color: #f8f8f8;
 +
        }
 +
 
 +
        #typora-quick-open-item {
 +
            background-color: #FAFAFA;
 +
            border-color: #FEFEFE #e5e5e5 #e5e5e5 #eee;
 +
            border-style: solid;
 +
            border-width: 1px;
 +
        }
 +
 
 +
        /** focus mode */
 +
        .on-focus-mode blockquote {
 +
            border-left-color: rgba(85, 85, 85, 0.12);
 +
        }
 +
 
 +
        header,
 +
        .context-menu,
 +
        .megamenu-content,
 +
        footer {
 +
            font-family: "Segoe UI", "Arial", sans-serif;
 +
        }
 +
 
 +
        .file-node-content:hover .file-node-icon,
 +
        .file-node-content:hover .file-node-open-state {
 +
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    <title>New-Project Design_1021</title>
 +
</head>
 +
 
 +
<body class='typora-export os-windows'>
 +
    <div class='typora-export-content'>
 +
        <div id='write' class=''>
 +
            <h1 id='project-design'><span>PROJECT DESIGN</span></h1>
 +
            <div class="section" id="section1"><h2 id='overview'><span>OVERVIEW</span></h2>
 +
            <p><span>Currently, the major disposal methods for PE are
 +
                </span><strong><span>incineration</span></strong><span> and
 +
                </span><strong><span>landfill</span></strong><span>, both of which are not the optimal way of disposing
 +
                    PE, for these two methods have led to </span><strong><span>negative environmental
 +
                        consequences</span></strong><span> not limited to the </span><strong><span>release of hazardous
 +
                        substances</span></strong><span>, and the </span><strong><span>occupancy of enormous land
 +
                        resources</span></strong><span>.</span></p>
 +
            <p><span>Therefore, we decided to take advantage of the power of nature, seeking
 +
                </span><strong><span>specific agents</span></strong><span> that possess the unique
 +
                </span><strong><span>ability of degrading PE</span></strong><span>, and further
 +
                </span><strong><span>modify</span></strong><span> and
 +
                </span><strong><span>optimize</span></strong><span> it to realize green and efficient degradation of
 +
                    PE.</span></p>
 +
            </div><div class="section" id="section2"><h2 id='to-determine-a-central-pe-degradation-element'><span>TO DETERMINE A CENTRAL PE DEGRADATION
 +
                    ELEMENT</span></h2>
 +
            <h3 id='agent-selection'><span>AGENT SELECTION</span></h3>
 +
            <p><span>During our preliminary stage of literature research, strains of microorganism as well as enzymes
 +
                    that both had the </span><strong><span>potential of PE degradation</span></strong><span> were
 +
                    obtained by us. An either-or decision must be made upon the selection of the PE-degrading agent.
 +
                    Without much hesitation, we selected </span><strong><span>enzymes</span></strong><span> instead of
 +
                    strains due to a more definite origin and characteristics provided by online databases. After
 +
                    screening through potential candidates, the very </span><strong><span>manganese
 +
                        peroxidase</span></strong><span> (MnP) was selected as our </span><strong><span>central
 +
                        functional element</span></strong><span>.</span></p>
 +
            <p><span>It is a highly glycosylated lignin peroxidase with heme</span><sup class="reference">[1,2]</span></sup><span>
 +
                    that can </span><strong><span>oxidize Mn</span><sup><span>2+</span></sup><span> to
 +
                        Mn</span><sup><span>3+</span></sup></strong><span>, the latter can be
 +
                </span><strong><span>chelated</span></strong><span> by ligands like </span><strong><span>oxalic
 +
                        acid</span></strong><span>, forming the
 +
                </span><strong><span>Mn</span><sup><span>3+</span></sup><span>-ligand chelate
 +
                        compound</span></strong><span> that can </span><strong><span>diffuse</span></strong><span>
 +
                    outside the enzyme for further degradation of lignin or other refractory
 +
                    chemicals</span><sup class="reference">[3]</span></sup><span>. </span></p>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/5/5e/T--CPU_CHINA--K3853008_Fig1.png"
 +
                    referrerpolicy="no-referrer" alt="T--CPU_CHINA--Design-1"></p>
 +
            <p><strong><span>Fig. 1 The catalytic cycle of MnP.</span></strong></p>
 +
            <p><span>It has been reported that </span><strong><span>MnP has a significant degradation efficiency on PE
 +
                        film</span></strong><span>. As reported before, the weight-average molecular weight (Mw) of PE
 +
                    was halved by MnP treatment for two days, showing its </span><strong><span>remarkable degradation
 +
                        efficacy</span></strong><sup class="reference">[4]</span></sup><span>. Thus, MnP was chosen by us as the key
 +
                    element for PE degradation.</span></p>
 +
            <h3 id='using-aao-as-a-better-approach-to-provide-substrate-for-mnp'><span>USING AAO AS A BETTER APPROACH TO
 +
                    PROVIDE SUBSTRATE FOR MnP</span></h3>
 +
            <p><span>It is shown on the catalytic cycle of MnP above that
 +
                </span><strong><span>H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub></strong><span>
 +
                    is required as its </span><strong><span>essential substrate</span></strong><span> for activating the
 +
                    enzymatic reaction. Yet an </span><strong><span>abnormally high</span></strong><span> concentration
 +
                    of H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span> could also
 +
                </span><strong><span>inhibit</span></strong><span>, even
 +
                </span><strong><span>deactivate</span></strong><span> the enzyme, which might happen when
 +
                    H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span> was added into the
 +
                    system manually and periodically. </span></p>
 +
            <p><span>Therefore, a more in-depth investigation was carried out to seek a solution. As a result, we
 +
                    discovered a specific type of enzyme, namely </span><strong><span>aryl alcohol oxidase
 +
                        (AAO)</span></strong><span>. It is an enzyme containing flavin-adenine-dinucleotide
 +
                    (FAD)</span><sup class="reference">[5]</span></sup><span> that catalyzes the oxidation of aromatic and aliphatic
 +
                    allylic primary alcohols (which are far less oxidative when compared to
 +
                    Mn</span><sup><span>3+</span></sup><span> and
 +
                    H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span>) to the corresponding
 +
                    aldehydes while </span><strong><span>reducing molecular oxygen to
 +
                        H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub></strong><sup class="reference">[6]</span></sup><span>.</span>
 +
            </p>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/e/e7/T--CPU_CHINA--BBa_K3853009_fig_1.png"
 +
                    referrerpolicy="no-referrer" alt="T--CPU_CHINA--Design-2"></p>
 +
            <p><strong><span>Fig. 2 The mechanism of AAO reducing molecular oxygen to
 +
                        H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span> by oxidizing
 +
                        4-methoxybenzyl alcohol.</span></strong><sup class="reference">[7]</span></sup></p>
 +
            <p><span>We learned from the literature</span><sup class="reference">[8]</span></sup><span> that AAO is able to produce
 +
                    H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span> in a
 +
                </span><strong><span>low but steady</span></strong><span> rate. Therefore, the
 +
                </span><strong><span>inhibition</span></strong><span> of MnP </span><strong><span>due
 +
                        to</span></strong><span> </span><strong><span>an</span></strong><span>
 +
                </span><strong><span>excess of
 +
                        H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub></strong><span>
 +
                    concentration can be </span><strong><span>effectively prevented</span></strong><span> when applying
 +
                    AAO as the source of H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span>.
 +
                    This would allow MnP to </span><strong><span>catalyze</span></strong><span> the PE-degrading
 +
                    reaction </span><strong><span>over a longer period of time</span></strong><span>, realizing a more
 +
                </span><strong><span>complete degradation</span></strong><span> of PE. In addition, since the two
 +
                    enzymes work in tandem, the </span><strong><span>cascade reaction</span></strong><span> mediated by
 +
                    the two can only be initiated when substrates of AAO is introduced to the system. Therefore, we can
 +
                </span><strong><span>achieve precise control</span></strong><span> to the onset and termination of the
 +
                    reactions via adding specific amount of substrates to the system in a given time, preventing
 +
                    uncontrollable situations from happening. As a result, we decided to select AAO as the assistant of
 +
                    MnP.</span></p>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/a/a4/T--CPU_CHINA--Design-3.jpg"
 +
                    referrerpolicy="no-referrer" alt="T--CPU_CHINA--Design-3"></p>
 +
            <p><strong><span>Fig. 3 The synergistic PE degradation effect of MnP and AAO.</span></strong></p>
 +
            </div><div class="section" id="section3"><h2 id='to-enhance-the-pe-degrading-efficiency-of-mnp'><span>TO ENHANCE THE PE-DEGRADING EFFICIENCY OF
 +
                    MnP</span></h2>
 +
            <h3 id='optimize-the-degradation-competence-of-mnp-by-directed-evolution'><span>OPTIMIZE THE DEGRADATION
 +
                    COMPETENCE OF MnP BY DIRECTED EVOLUTION</span></h3>
 +
            <p><span>As our key PE-degrading enzyme, manganese peroxidase (MnP) undertakes a
 +
                </span><strong><span>fundamental</span></strong><span> role of inflicting oxidation to PE by
 +
                    continuously producing Mn</span><sup><span>3+</span></sup><span> ions. Therefore,
 +
                </span><strong><span>enhancing</span></strong><span> the degradation
 +
                </span><strong><span>efficiency</span></strong><span> of MnP is beneficial to reach
 +
                </span><strong><span>a more complete destruction</span></strong><span> of PE films.</span></p>
 +
            <p><span>In theory, there are </span><strong><span>two approaches</span></strong><span> of reinforce the
 +
                    degradation efficacy of MnP, whether by </span><strong><span>increasing the
 +
                        activity</span></strong><span> of MnP to realize a stronger oxidative capacity, or by
 +
                </span><strong><span>improving the stability</span></strong><span> of MnP to prolong its duration of
 +
                    effect. However, since the substrate and catalysate of MnP are both highly-oxidative, simply
 +
                    increasing its activity without restrictions is bound to cause irreversible harm not only to the MnP
 +
                    itself, but also to other affiliated elements in our design, AAO for instance. Therefore, we decided
 +
                    to </span><strong><span>improve the stability</span></strong><span> of MnP by proposing a
 +
                </span><strong><span>semi-rational directed evolution
 +
                        strategy</span></strong><sup class="reference">[9]</span></sup><span>towards it, with the hope to increase
 +
                    its tolerance of high temperature, acidic pH, as well as different types of organic solvents, all of
 +
                    which are common inhibitory physiochemical properties that may severely impact the activity of
 +
                    MnP.</span></p>
 +
            <p><span>For the results of our directed-evolution attempt, see
 +
                </span><strong><em><span>Improvement</span></em><span> (链接)</span></strong><span> page</span></p>
 +
            <h3 id='facilitate-the-surface-adherence-of-mnp-by-introducing-hfb1'><span>FACILITATE THE SURFACE ADHERENCE
 +
                    OF MnP BY INTRODUCING HFB1</span></h3>
 +
            <p><span>Back to the stage where we were searching for agents with PE degradation efficacy, we noticed that
 +
                    certain bacterial or fungal strains capable of degrading PE could produce
 +
                </span><strong><span>biosurfactant</span></strong><span> to assist their adherence and colonization on
 +
                    the hydrophobic surface of plastics, so that they could degrade PE in a faster pace. This inspired
 +
                    us to introduce biosurfactant into our design, aiming to </span><strong><span>increase the
 +
                        hydrophilicity</span></strong><span> of the surface of PE. </span></p>
 +
            <p><span>As a result, our focus was concentrated on hydrophobin-1 (HFB1), a kind of class Ⅱ HFBs derived
 +
                    from </span><em><span>Trichoderma reesei</span></em><sup class="reference">[38]</span></sup><span>. It is rich
 +
                    in hydrophobic amino acids, endowing its surface activity. By
 +
                </span><strong><span>self-assembling</span></strong><span> at hydrophilic-hydrophobic interfaces
 +
                    autonomously, HFB1 can </span><strong><span>enhance the affinity</span></strong><span> between
 +
                    hydrophilic proteins and hydrophobic materials</span><sup class="reference">[11]</span></sup><strong><span>such
 +
                        as PE</span></strong><span>, thus facilitating its contact with aqueous environment, thereby
 +
                    facilitating MnP to degrade PE. </span></p>
 +
            <p><span>What&#39;s more, compared with other members of HFBs, HFB1 has </span><strong><span>better
 +
                        stability</span></strong><span> and </span><strong><span>higher surface
 +
                        activity</span></strong><span>, which means it can maintain its function of adherence on
 +
                    hydrophobic substances </span><strong><span>more firmly</span></strong><span> for a
 +
                </span><strong><span>longer period of time</span></strong><span>.</span></p>
 +
            <p><span>Therefore, in our project, HFB1 is selected and used as a biosurfactant to produce consistent
 +
                    surface activity on PE, thereby promoting the adherence of MnP on PE surface, which helps to
 +
                </span><strong><span>improve</span></strong><span> the degradation
 +
                </span><strong><span>efficacy</span></strong><span> of this enzyme.</span></p>
 +
            </div><div class="section" id="section4"><h2 id='to-converge-the-advantages-of-three-functional-proteins'><span>TO CONVERGE THE ADVANTAGES OF THREE
 +
                    FUNCTIONAL PROTEINS</span></h2>
 +
            <p><span>Now that the three functional proteins were selected, all of which possesses individual functions
 +
                    that could contribute to the degradation of PE, instead of directly applying all of them by simply
 +
                    adding them into the system separately, we began to consider the possibility of
 +
                </span><strong><span>combining</span></strong><span> these discrete parts into a
 +
                </span><strong><span>composite entity</span></strong><span>, enabling the production of a strong
 +
                    synergistic effect which may lead to an significant improvement on efficacy. </span></p>
 +
            <h3 id='getting-closer-to-the-surface-of-pe'><span>GETTING CLOSER TO THE SURFACE OF PE</span></h3>
 +
            <p><span>The first idea that struck us was that we could minimize the spatial distance between MnP and PE by
 +
                </span><strong><span>fusing HFB1</span></strong><span> on the enzyme. Similar strategy could also be
 +
                    applied on AAO to generate fusion protein as well. In this way, our functional enzymes can
 +
                    simultaneously be anchored to the PE surface with the aid of fused HFB1, so that the
 +
                </span><strong><span>diffusion distance</span></strong><span> of
 +
                    Mn</span><sup><span>3+</span></sup><span>-ligand chelate compound towards PE could be
 +
                </span><strong><span>significantly lessened</span></strong><span>, enabling a more efficient degradation
 +
                    outcome. Meanwhile, the
 +
                    H</span><sub><span>2</span></sub><span>O</span><sub><span>2</span></sub><span> generated by AAO can
 +
                    also become more accessible to MnP when the two enzymes are </span><strong><span>closely
 +
                        anchored</span></strong><span> to the surface of PE.</span></p>
 +
            <p><span>Therefore, we delved into literatures and previous iGEM projects to look for ideal solutions. It
 +
                    turned out that there existed a versatile protein ligation system, i.e.
 +
                </span><strong><span>SpyCatcher/SpyTag system</span></strong><sup class="reference">[12,13]</span></sup><span>, that
 +
                    has been widely adopted by many laboratories and iGEM teams for construction of multi-domain
 +
                    protein. This system contains </span><strong><span>two essential elements</span></strong><span>:
 +
                </span></p>
 +
            <ul>
 +
                <li><strong><span>SpyCatcher:</span></strong><span> a modified immunoglobulin-like domain CnaB2 from a
 +
                    </span><em><span>Streptococcus pyogenes</span></em><span> surface protein</span></li>
 +
                <li><strong><span>SpyTag:</span></strong><span> a cognate 13-amino-acid peptide</span></li>
 +
            </ul>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/b/b6/T--CPU_CHINA--Design-4.1.png"
 +
                    referrerpolicy="no-referrer" alt="T--CPU_CHINA--Design-4"></p>
 +
            <p><strong><span>Fig. 4 The isopepide-forming mechanism between the two Spy domains.</span></strong><span>
 +
                </span><em><span>Glu77 &amp; Lys31 are the residues on SpyCatcher; Asp117 is the residue on
 +
                        SpyTag.</span></em></p>
 +
            <p><span>The two domains can </span><strong><span>autonomously form a covalent isopeptide
 +
                        bond</span></strong><span> between each other, thereby linking the two portions together. By
 +
                    linking the Spy domains on the N-terminal or C-terminal of the target protein with
 +
                </span><strong><span>elastin-like protein</span></strong><span> (ELP) or
 +
                </span><strong><span>serine/glycine link</span></strong><span> (Ser/Gly
 +
                    link)</span><sup class="reference">[14]</span></sup><span>, its structure and function are generally
 +
                </span><strong><span>unaffected</span></strong><span>, while the formation of isopeptide bond between
 +
                    SpyCatcher and SpyTag remains effective and efficient. By adopting this system, MnP and AAO that was
 +
                    fused with HFB1 are able to stick to surface of PE, realizing a better spatial concentration on
 +
                    it.</span></p>
 +
            <h3 id='getting-closer-with-each-other'><span>GETTING CLOSER WITH EACH OTHER</span></h3>
 +
            <p><span>Albeit introducing SpyCatcher/SpyTag connect system into our design could have a positive influence
 +
                    on accelerating PE degradation, several shortcomings are not yet solved. For example, the adherence
 +
                    of MnP-HFB1 and AAO-HFB1 fusion proteins on the surface of PE are likely to be unordered instead of
 +
                    evenly distributed. Protein clusters of the same type of fusion protein are likely to be formed on
 +
                    the PE surface, preventing thorough substance exchange between discrete protein molecules. Also, the
 +
                    maintenance of optimum functioning ratio between MnP and AAO cannot be guaranteed due to the
 +
                    arbitrary distribution on the PE surface. Both of the two uncontrollable conditions will reduce the
 +
                    efficacy of PE degradation.</span></p>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/7/7e/T--CPU_CHINA--Design-5.PNG"
 +
                    referrerpolicy="no-referrer" alt="T--CPU_CHINA--Design-5"></p>
 +
            <p><strong><span>Fig. 5 The potentially huge differences between ideality and reality.</span></strong></p>
 +
            <p><span>To enable the binding of MnP and AAO on the surface of PE in a more organized manner, we adopted a
 +
                    recently reported CRISPR/Cas-based DNA anchoring system</span><sup class="reference">[15]</span></sup><span> to
 +
                    our design. This system utilizes an deactivated CRISPR-associated protein 9 linked to a SpyCatcher
 +
                    domain (dCas9-SpyCatcher), which can not only form a covalent bond with proteins fused with SpyTag
 +
                    domain, but also recognize and bind to complementary DNA sequences after incorporating a
 +
                    single-guide RNA (sgRNA) without cleavage activity.</span>
 +
                <span>Therefore, by specially designing a double-stranded DNA (dsDNA) with multiple sequence segments
 +
                    complementary to different sgRNAs, the dCas9-SpyCatcher incorporated with different types of sgRNAs
 +
                    and functional proteins can be anchored to the double-stranded DNA in a predetermined number and
 +
                    proportion. </span>
 +
            </p>
 +
            <p><span>In our eventual design, the three functional proteins are all fused with SpyTag, covalently linked
 +
                    with dCas9-SpyCatcher, and anchored to the same dsDNA. In this way, the spatial distance between MnP
 +
                    and AAO could be remarkably reduced. The close proximity and determined proportion between the two
 +
                    enzymes can greatly facilitate substance exchange, thereby releasing a steady flow of PE-oxidizing
 +
                    agent when given sufficient substrate. Moreover, instead of pulling one individual enzyme once for
 +
                    all, HFB1, or HFB1s, can now paste the whole protein-nucleic-acid complex onto the surface of PE
 +
                    synergistically. </span></p>
 +
            </div><div class="section" id="section5"><h2 id='the-overall-diagram'><span>THE OVERALL DIAGRAM</span></h2>
 +
            <p><span>Eventually, by combining the three PE-degrading elements with the two assembly systems, we are able
 +
                     to construct a new type of protein-nucleic-acid complex that possesses an enhanced ability for PE
 +
                    degradation. And we name it, polyethylene degradist.</span></p>
 +
            <p><span>A demonstrative graph is shown below. </span></p>
 +
            <p><img src="https://static.igem.org/mediawiki/2021/e/ee/T--CPU_CHINA--Design-6.jpg"
 +
                    referrerpolicy="no-referrer" alt="Fig6"></p>
 +
            <p><strong><span>Fig. 6 The final conceptual design overview of our PE-degrading system.</span></strong></p>
 +
            <p>&nbsp;</p>
 
             <h4 class="mume-header" id="references">References</h4>
 
             <h4 class="mume-header" id="references">References</h4>
 
             <p class="reference">[1] Martinez A T, RuizMartinez A T, Ruiz--Duenas F J, Camarero S, et al. Oxidoreductases on their
 
             <p class="reference">[1] Martinez A T, RuizMartinez A T, Ruiz--Duenas F J, Camarero S, et al. Oxidoreductases on their
Line 440: Line 1,734:
 
                     complexes. </span><em><span>Chem Commun (Camb)</span></em><span>. 2020;56(36):4950-4953.
 
                     complexes. </span><em><span>Chem Commun (Camb)</span></em><span>. 2020;56(36):4950-4953.
 
                     doi:10.1039/d0cc01174f</span></p>
 
                     doi:10.1039/d0cc01174f</span></p>
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Revision as of 00:05, 22 October 2021

<!doctype html> New-Project Design_1021

PROJECT DESIGN

OVERVIEW

Currently, the major disposal methods for PE are incineration and landfill, both of which are not the optimal way of disposing PE, for these two methods have led to negative environmental consequences not limited to the release of hazardous substances, and the occupancy of enormous land resources.

Therefore, we decided to take advantage of the power of nature, seeking specific agents that possess the unique ability of degrading PE, and further modify and optimize it to realize green and efficient degradation of PE.

TO DETERMINE A CENTRAL PE DEGRADATION ELEMENT

AGENT SELECTION

During our preliminary stage of literature research, strains of microorganism as well as enzymes that both had the potential of PE degradation were obtained by us. An either-or decision must be made upon the selection of the PE-degrading agent. Without much hesitation, we selected enzymes instead of strains due to a more definite origin and characteristics provided by online databases. After screening through potential candidates, the very manganese peroxidase (MnP) was selected as our central functional element.

It is a highly glycosylated lignin peroxidase with heme[1,2] that can oxidize Mn2+ to Mn3+, the latter can be chelated by ligands like oxalic acid, forming the Mn3+-ligand chelate compound that can diffuse outside the enzyme for further degradation of lignin or other refractory chemicals[3].

T--CPU_CHINA--Design-1

Fig. 1 The catalytic cycle of MnP.

It has been reported that MnP has a significant degradation efficiency on PE film. As reported before, the weight-average molecular weight (Mw) of PE was halved by MnP treatment for two days, showing its remarkable degradation efficacy[4]. Thus, MnP was chosen by us as the key element for PE degradation.

USING AAO AS A BETTER APPROACH TO PROVIDE SUBSTRATE FOR MnP

It is shown on the catalytic cycle of MnP above that H2O2 is required as its essential substrate for activating the enzymatic reaction. Yet an abnormally high concentration of H2O2 could also inhibit, even deactivate the enzyme, which might happen when H2O2 was added into the system manually and periodically.

Therefore, a more in-depth investigation was carried out to seek a solution. As a result, we discovered a specific type of enzyme, namely aryl alcohol oxidase (AAO). It is an enzyme containing flavin-adenine-dinucleotide (FAD)[5] that catalyzes the oxidation of aromatic and aliphatic allylic primary alcohols (which are far less oxidative when compared to Mn3+ and H2O2) to the corresponding aldehydes while reducing molecular oxygen to H2O2[6].

T--CPU_CHINA--Design-2

Fig. 2 The mechanism of AAO reducing molecular oxygen to H2O2 by oxidizing 4-methoxybenzyl alcohol.[7]

We learned from the literature[8] that AAO is able to produce H2O2 in a low but steady rate. Therefore, the inhibition of MnP due to an excess of H2O2 concentration can be effectively prevented when applying AAO as the source of H2O2. This would allow MnP to catalyze the PE-degrading reaction over a longer period of time, realizing a more complete degradation of PE. In addition, since the two enzymes work in tandem, the cascade reaction mediated by the two can only be initiated when substrates of AAO is introduced to the system. Therefore, we can achieve precise control to the onset and termination of the reactions via adding specific amount of substrates to the system in a given time, preventing uncontrollable situations from happening. As a result, we decided to select AAO as the assistant of MnP.

T--CPU_CHINA--Design-3

Fig. 3 The synergistic PE degradation effect of MnP and AAO.

TO ENHANCE THE PE-DEGRADING EFFICIENCY OF MnP

OPTIMIZE THE DEGRADATION COMPETENCE OF MnP BY DIRECTED EVOLUTION

As our key PE-degrading enzyme, manganese peroxidase (MnP) undertakes a fundamental role of inflicting oxidation to PE by continuously producing Mn3+ ions. Therefore, enhancing the degradation efficiency of MnP is beneficial to reach a more complete destruction of PE films.

In theory, there are two approaches of reinforce the degradation efficacy of MnP, whether by increasing the activity of MnP to realize a stronger oxidative capacity, or by improving the stability of MnP to prolong its duration of effect. However, since the substrate and catalysate of MnP are both highly-oxidative, simply increasing its activity without restrictions is bound to cause irreversible harm not only to the MnP itself, but also to other affiliated elements in our design, AAO for instance. Therefore, we decided to improve the stability of MnP by proposing a semi-rational directed evolution strategy[9]towards it, with the hope to increase its tolerance of high temperature, acidic pH, as well as different types of organic solvents, all of which are common inhibitory physiochemical properties that may severely impact the activity of MnP.

For the results of our directed-evolution attempt, see Improvement (链接) page

FACILITATE THE SURFACE ADHERENCE OF MnP BY INTRODUCING HFB1

Back to the stage where we were searching for agents with PE degradation efficacy, we noticed that certain bacterial or fungal strains capable of degrading PE could produce biosurfactant to assist their adherence and colonization on the hydrophobic surface of plastics, so that they could degrade PE in a faster pace. This inspired us to introduce biosurfactant into our design, aiming to increase the hydrophilicity of the surface of PE.

As a result, our focus was concentrated on hydrophobin-1 (HFB1), a kind of class Ⅱ HFBs derived from Trichoderma reesei[38]. It is rich in hydrophobic amino acids, endowing its surface activity. By self-assembling at hydrophilic-hydrophobic interfaces autonomously, HFB1 can enhance the affinity between hydrophilic proteins and hydrophobic materials[11]such as PE, thus facilitating its contact with aqueous environment, thereby facilitating MnP to degrade PE.

What's more, compared with other members of HFBs, HFB1 has better stability and higher surface activity, which means it can maintain its function of adherence on hydrophobic substances more firmly for a longer period of time.

Therefore, in our project, HFB1 is selected and used as a biosurfactant to produce consistent surface activity on PE, thereby promoting the adherence of MnP on PE surface, which helps to improve the degradation efficacy of this enzyme.

TO CONVERGE THE ADVANTAGES OF THREE FUNCTIONAL PROTEINS

Now that the three functional proteins were selected, all of which possesses individual functions that could contribute to the degradation of PE, instead of directly applying all of them by simply adding them into the system separately, we began to consider the possibility of combining these discrete parts into a composite entity, enabling the production of a strong synergistic effect which may lead to an significant improvement on efficacy.

GETTING CLOSER TO THE SURFACE OF PE

The first idea that struck us was that we could minimize the spatial distance between MnP and PE by fusing HFB1 on the enzyme. Similar strategy could also be applied on AAO to generate fusion protein as well. In this way, our functional enzymes can simultaneously be anchored to the PE surface with the aid of fused HFB1, so that the diffusion distance of Mn3+-ligand chelate compound towards PE could be significantly lessened, enabling a more efficient degradation outcome. Meanwhile, the H2O2 generated by AAO can also become more accessible to MnP when the two enzymes are closely anchored to the surface of PE.

Therefore, we delved into literatures and previous iGEM projects to look for ideal solutions. It turned out that there existed a versatile protein ligation system, i.e. SpyCatcher/SpyTag system[12,13], that has been widely adopted by many laboratories and iGEM teams for construction of multi-domain protein. This system contains two essential elements:

  • SpyCatcher: a modified immunoglobulin-like domain CnaB2 from a Streptococcus pyogenes surface protein
  • SpyTag: a cognate 13-amino-acid peptide

T--CPU_CHINA--Design-4

Fig. 4 The isopepide-forming mechanism between the two Spy domains. Glu77 & Lys31 are the residues on SpyCatcher; Asp117 is the residue on SpyTag.

The two domains can autonomously form a covalent isopeptide bond between each other, thereby linking the two portions together. By linking the Spy domains on the N-terminal or C-terminal of the target protein with elastin-like protein (ELP) or serine/glycine link (Ser/Gly link)[14], its structure and function are generally unaffected, while the formation of isopeptide bond between SpyCatcher and SpyTag remains effective and efficient. By adopting this system, MnP and AAO that was fused with HFB1 are able to stick to surface of PE, realizing a better spatial concentration on it.

GETTING CLOSER WITH EACH OTHER

Albeit introducing SpyCatcher/SpyTag connect system into our design could have a positive influence on accelerating PE degradation, several shortcomings are not yet solved. For example, the adherence of MnP-HFB1 and AAO-HFB1 fusion proteins on the surface of PE are likely to be unordered instead of evenly distributed. Protein clusters of the same type of fusion protein are likely to be formed on the PE surface, preventing thorough substance exchange between discrete protein molecules. Also, the maintenance of optimum functioning ratio between MnP and AAO cannot be guaranteed due to the arbitrary distribution on the PE surface. Both of the two uncontrollable conditions will reduce the efficacy of PE degradation.

T--CPU_CHINA--Design-5

Fig. 5 The potentially huge differences between ideality and reality.

To enable the binding of MnP and AAO on the surface of PE in a more organized manner, we adopted a recently reported CRISPR/Cas-based DNA anchoring system[15] to our design. This system utilizes an deactivated CRISPR-associated protein 9 linked to a SpyCatcher domain (dCas9-SpyCatcher), which can not only form a covalent bond with proteins fused with SpyTag domain, but also recognize and bind to complementary DNA sequences after incorporating a single-guide RNA (sgRNA) without cleavage activity. Therefore, by specially designing a double-stranded DNA (dsDNA) with multiple sequence segments complementary to different sgRNAs, the dCas9-SpyCatcher incorporated with different types of sgRNAs and functional proteins can be anchored to the double-stranded DNA in a predetermined number and proportion.

In our eventual design, the three functional proteins are all fused with SpyTag, covalently linked with dCas9-SpyCatcher, and anchored to the same dsDNA. In this way, the spatial distance between MnP and AAO could be remarkably reduced. The close proximity and determined proportion between the two enzymes can greatly facilitate substance exchange, thereby releasing a steady flow of PE-oxidizing agent when given sufficient substrate. Moreover, instead of pulling one individual enzyme once for all, HFB1, or HFB1s, can now paste the whole protein-nucleic-acid complex onto the surface of PE synergistically.

THE OVERALL DIAGRAM

Eventually, by combining the three PE-degrading elements with the two assembly systems, we are able to construct a new type of protein-nucleic-acid complex that possesses an enhanced ability for PE degradation. And we name it, polyethylene degradist.

A demonstrative graph is shown below.

Fig6

Fig. 6 The final conceptual design overview of our PE-degrading system.

 

References

[1] Martinez A T, RuizMartinez A T, Ruiz--Duenas F J, Camarero S, et al. Oxidoreductases on their way to industrial biotransformations[J]. Biotechnology Advances, 2017, 35(6): 81535(6): 815--831.831.

[2] Chandra R, Kumar, V., Yadav, S. Extremophilic Enzymatic Processing of Lignocellulosic FChandra R, Kumar, V., Yadav, S. Extremophilic Enzymatic Processing of Lignocellulosic Feedstocks to Bioenergy[M]. Springer International Publishing, 2017

[3] Saez--Jimenez V, Baratto M C, Pogni R, et al. Demonstration of LigninJimenez V, Baratto M C, Pogni R, et al. Demonstration of Lignin--toto--Peroxidase DirePeroxidase Direct Electron Transfer A TRANSIENT--STATE KINETICS, DIRECTED MUTAGENESIS, EPR, ASTATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY[J]. Journal of Biological Chemistry, 2015, 290(38): 23201--23213.23213

[4] Iiyoshi Y, Tsutsumi Y, Nishida T. Polyethylene degradation by ligninida T. Polyethylene degradation by lignin--degrading fungi and degrading fungi and managanese peroxidase[J]. Journal of Wood Science, 1998, 44(3): 222--229.229.

[5] Farmer V C, Henderson M E, Russell J D. Aromatic alcohol oxidase activity in the growth medium of Polystictus versic olor[J]. The Biochemical journal, 1960, 74: 257 62.

[6] Ruiz Duenas F J, Martinez A T. Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how w e can take advantage of this[J]. Microbial Biotechnology, 2009, 2(2): 164 177.

[7] Serrano A, Carro J, Martinez A T. Reaction mechanisms and applications of aryl alcohol oxidase[J]. The Enzymes, 2020, 47: 167 192.

[8] Sugano Y, Matsushima Y, Shoda M. Complete decolorization of the anthraquinone dye Reactive blue 5 by the concerted action of two peroxidases from Thanatephorus cucumeris Dec 1. Appl Microbiol Biotechnol. 2006;73(4):862-871. doi:10.1007/s00253-006-0545-9

[9] Steiner K, Schwab H. Recent advances in rational approaches for enzyme engineering[J]. Computational and structural biotechnology journal, 2012, 2: e201209010.

[10] Nakari T, Alatalo E, Penttila M E. ISOLATION OF TRICHODERMA REESEI GENES HIGHLY EXPRESSED ON GLUCOSE CONTAINING MEDIA CHARACTERIZATION OF THE TEF1 GENE ENCODING TRANSLATION ELONGATI ON FACTOR 1 ALPHA[J]. Gene, 1993, 136(1 2): 313 318.

[11] Linder M B, Szilvay G R, Nakari Setala T, et al. Hydrophobins: the protein amphiphiles of filamentous fungi[J]. Fems Microbiology Reviews, 2005, 29(5): 877 896.

[12] Kang H J, Baker E N. Intramolecular isopeptide bonds: protein crosslinks built for stress? [J]. Trends in biochemical sciences, 2011, 36(4): 229 237.

[13] Hagan R M, Björnsson R, Mcmahon S A, et al. NMR spectroscopic and theoretical analysis of a spontaneously formed Lys Asp isopeptide bond[J]. Angewandte Chemie (International ed. in English), 2010, 49(45): 8421 8425.

[14] Reddington SC, Howarth M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 2015;29:94-99. doi:10.1016/j.cbpa.2015.10.002

[15] Lim S, Kim J, Kim Y, Xu D, Clark DS. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chem Commun (Camb). 2020;56(36):4950-4953. doi:10.1039/d0cc01174f