Difference between revisions of "Team:NUDT CHINA/Model"
Zhuchushu13 (Talk | contribs) |
Zhuchushu13 (Talk | contribs) |
||
| (43 intermediate revisions by 2 users not shown) | |||
| Line 69: | Line 69: | ||
<meta name="HandheldFriendly" content="True" /> | <meta name="HandheldFriendly" content="True" /> | ||
<meta name="MobileOptimized" content="320" /> | <meta name="MobileOptimized" content="320" /> | ||
| − | + | <meta name="viewport" content="width=device-width, initial-scale=1.0",minimum-scale=0.2, maximum-scale=2.0, user-scalable=yes" /> | |
<meta content="on" http-equiv="cleartype" /> | <meta content="on" http-equiv="cleartype" /> | ||
<meta property="og:title" content="Home" /> | <meta property="og:title" content="Home" /> | ||
| Line 88: | Line 88: | ||
<div class="inner-wrap"> | <div class="inner-wrap"> | ||
<!-- Off Canvas Menu --> | <!-- Off Canvas Menu --> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- close the off-canvas menu --> | <!-- close the off-canvas menu --> | ||
<a class="exit-off-canvas"></a> | <a class="exit-off-canvas"></a> | ||
| Line 152: | Line 115: | ||
<a class="nav-global__link" title="" href="#">AWARDS</a></li> | <a class="nav-global__link" title="" href="#">AWARDS</a></li> | ||
</ul> | </ul> | ||
| − | <style>#menu06{width:200px;right:0px;} #menu07{width:200px;right:0px;} | + | <style> |
| + | .content{ | ||
| + | box-shadow: 6px 6px 3px #888888; | ||
| + | } | ||
| + | #menu06{width:200px;right:0px;} #menu07{width:200px;right:0px;} | ||
#menu01 .arrow-up{margin-right:6.429rem;} | #menu01 .arrow-up{margin-right:6.429rem;} | ||
#menu02 .arrow-up{margin-right:90px;} #menu03 .arrow-up{margin-right:90px;} #menu06 .arrow-up{margin-right:120px;} #menu07 .arrow-up{margin-right:27px;} #menu01{width:200px;right:31rem;} #menu02{width:200px;right:27rem;} #menu03{width:200px;right:21.3rem;} | #menu02 .arrow-up{margin-right:90px;} #menu03 .arrow-up{margin-right:90px;} #menu06 .arrow-up{margin-right:120px;} #menu07 .arrow-up{margin-right:27px;} #menu01{width:200px;right:31rem;} #menu02{width:200px;right:27rem;} #menu03{width:200px;right:21.3rem;} | ||
| Line 322: | Line 289: | ||
</div> | </div> | ||
</nav> | </nav> | ||
| − | <style>.nav-global ul li a{ font-size:0.85rem; } @media screen and (max-width: 865px) { .nav-global ul li a{ font-size:0.85rem; } }</style> | + | <style>.nav-global ul li a{ font-size:0.85rem; } @media screen and (max-width: 865px) { .nav-global ul li a{ font-size:0.85rem; } } |
| + | |||
| + | #feature{ | ||
| + | background-size:100%; | ||
| + | } | ||
| + | @media screen and (max-width: 1200px) { | ||
| + | #feature{ | ||
| + | background-size:1200px; | ||
| + | } | ||
| + | } | ||
| + | </style> | ||
<div class="breadcrumb-share-wrap"> | <div class="breadcrumb-share-wrap"> | ||
| Line 335: | Line 312: | ||
<!-- end .row --></div> | <!-- end .row --></div> | ||
<!-- end #header-outer-wrap--> | <!-- end #header-outer-wrap--> | ||
| − | <div id="feature" class="row collapse text-light feature-footer-active"> | + | <div id="feature" class="row collapse text-light feature-footer-active" style="background-color: rgb(3,13,38);background-image:url(https://static.igem.org/mediawiki/2016/thumb/3/3f/NUDT_CHINA2016_FILES-img-banner-3.0.png/800px-NUDT_CHINA2016_FILES-img-banner-3.0.png);background-position:0px -350px;background-repeat:no-repeat;"> |
<div class="inner-wrap medium-24 columns"> | <div class="inner-wrap medium-24 columns"> | ||
<!--TYPO3SEARCH_begin--> | <!--TYPO3SEARCH_begin--> | ||
| Line 341: | Line 318: | ||
<div class="carousel " id="slideshow-4811" data-cycle-log="false" data-cycle-auto-height="calc" data-cycle-prev="#prev-4811" data-cycle-next="#next-4811" data-cycle-pager="#cycle-pager-4811" data-cycle-easing="easeInOutQuad" data-cycle-fx="scrollVertUp" data-cycle-center-horz="true" data-cycle-speed="1000" data-cycle-timeout="8000" data-cycle-paused="true" data-cycle-slides="> div.slide"> | <div class="carousel " id="slideshow-4811" data-cycle-log="false" data-cycle-auto-height="calc" data-cycle-prev="#prev-4811" data-cycle-next="#next-4811" data-cycle-pager="#cycle-pager-4811" data-cycle-easing="easeInOutQuad" data-cycle-fx="scrollVertUp" data-cycle-center-horz="true" data-cycle-speed="1000" data-cycle-timeout="8000" data-cycle-paused="true" data-cycle-slides="> div.slide"> | ||
<div class="slide first"> | <div class="slide first"> | ||
| − | <link rel="stylesheet" href="/Team | + | <link rel="stylesheet" href="/Team--NUDT_CHINA/CSS3.css" /> |
<!-- Quick initialize with theme color from first slide --> | <!-- Quick initialize with theme color from first slide --> | ||
<div class="row collapse " id="c6601" data-theme-class="bh-color__theme-c6601"> | <div class="row collapse " id="c6601" data-theme-class="bh-color__theme-c6601"> | ||
<div class="columns"> | <div class="columns"> | ||
| − | <section class="big-header bh__height-size-3 text-light bh__video"> | + | <section class="big-header bh__height-size-3 text-light bh__video" style="height:300px;"> |
| − | <div class="bh__background bh__background-color | + | <div class="bh__background bh__background-color "></div> |
| − | <div class="bh__content-wrap"> | + | <div class="bh__content-wrap" style="height:120px;"> |
<div class="bh__content "> | <div class="bh__content "> | ||
<div class="bh__content-inner-wrap"> | <div class="bh__content-inner-wrap"> | ||
<!--h3 class="bh__subtitle"><a href="http#/explore/whats-here/exhibits/brick-by-brick/"><span class="line-wrap"><span class="line"><span class="bh__info">Exhibit / </span>Brick by Brick</span></span></a></h3--> | <!--h3 class="bh__subtitle"><a href="http#/explore/whats-here/exhibits/brick-by-brick/"><span class="line-wrap"><span class="line"><span class="bh__info">Exhibit / </span>Brick by Brick</span></span></a></h3--> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<h2 style="font-size:36px;" class="bh__title "> | <h2 style="font-size:36px;" class="bh__title "> | ||
<!--<a href="#">--> | <!--<a href="#">--> | ||
| − | + | <span style="line-height:46px;" class="line-wrap"> | |
| − | + | <span style="line-height:46px;" class="line">Development of A Novel</span></span> | |
| − | + | <span style="line-height:46px;" class="line-wrap"> | |
| − | + | <span style="line-height:46px;" class="line">Blood-MicroRNA Handy Detection System with CRISPR</span></span> | |
| − | <!--</a>--> | + | <!--</a>--></h2> |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 378: | Line 345: | ||
<div class="cycle-pager" id="cycle-pager-4811"></div> | <div class="cycle-pager" id="cycle-pager-4811"></div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
<!-- end #feature --> | <!-- end #feature --> | ||
<!-- end .columns --></div> | <!-- end .columns --></div> | ||
| − | <div style="margin-top: | + | <div style="margin-top:20px;" id="content-wrap-ink"><!-- adjust in add_JS --> |
| − | <div class="row footer-link" style=""> | + | <div class="row footer-link" style=""> |
| + | <div style="text-align:right;"><h5> | ||
| + | <a style="color:rgb(10,31,84);" href="/Team:NUDT_CHINA">HomePage</a> • | ||
| + | <!-- 修改这里！！ -->AWARDS • | ||
| + | <a style="color:rgb(10,31,84);" href="/Team:NUDT_CHINA/Model"><!-- 修改这里！！ -->Model</a> | ||
| + | </h5><hr style="width:40%;margin-left:60%;border-top:1px solid rgb(10,31,84);" /></div> | ||
| Line 392: | Line 364: | ||
| − | + | ||
| − | + | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <!--标题--> | ||
<h2> | <h2> | ||
<span><span style="color:#7f1015">Abstract</span></span><hr /> | <span><span style="color:#7f1015">Abstract</span></span><hr /> | ||
| Line 402: | Line 386: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">This | <span style="line-height:2;font-family:Perpetua;font-size:18px;">This | ||
| − | model | + | model is created to evaluate the effectiveness of initial design, and offers |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | guidelines on how the system can (or must) be improved. (You can go to <a href="/Team:NUDT_CHINA/Design">PROJECT. page</a> to see more.) The core idea is to simulate the process of producing the signal which can be detected, and draw a conclusion by obtaining the relationship between the signal intensity and the concentration of miRNA. | ||
</span> | </span> | ||
</p> | </p> | ||
| Line 420: | Line 397: | ||
<span><span style="color:#7f1015">Introduction </span></span><hr /> | <span><span style="color:#7f1015">Introduction </span></span><hr /> | ||
</h2> | </h2> | ||
| − | |||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">We |
| − | RCA model and a | + | create mathematical models of two aspects of our project, a RCA model and a |
| − | </span> | + | signal detection model.</span> |
</p> | </p> | ||
</br> | </br> | ||
| − | |||
| Line 439: | Line 414: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">The RCA | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The RCA | ||
| − | model | + | model is based on the Michaelis-Menten equation. We can obtain the relationship |
| − | concentration of miRNA and the number of stem-loop structures | + | between the concentration of miRNA and the number of stem-loop structures |
| − | through theoretical calculation, and our experimental results | + | through theoretical calculation, and we use our experimental results to |
calculate the parameters we introduced previously.</span> | calculate the parameters we introduced previously.</span> | ||
| Line 452: | Line 427: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">In the |
| − | signal detection model | + | signal detection model, we combine a probability model with the kinetic equation |
| − | + | of enzymatic reaction, so we can obtain the relationship between the number of | |
stem-loop structures and the signal intensity under the premise of adding a | stem-loop structures and the signal intensity under the premise of adding a | ||
| − | certain amount of fusion proteins of dCas9 and split-HRP fragments | + | certain amount of the fusion proteins of dCas9 and split-HRP fragments. </span> |
| − | + | ||
| − | </span> | + | |
</p> | </p> | ||
</br> | </br> | ||
| Line 471: | Line 444: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">By | <span style="line-height:2;font-family:Perpetua;font-size:18px;">By | ||
| − | integrating the two models, the impacts of the | + | integrating the two models, we can theoretically predict the impacts of the |
| − | the signal to noise ratio | + | molecule number of proteins on the signal to noise ratio and explain the trend |
| − | signal intensity | + | of the signal intensity variation against the change of the concentration of miRNA in our |
| − | work | + | wet-lab work. |
| + | </span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 491: | Line 465: | ||
<!--标题--> | <!--标题--> | ||
<h3> | <h3> | ||
| − | <span><span style="color:#7f1015"> About model </span></span | + | <span><span style="color:#7f1015"> About model </span></span> |
</h3> | </h3> | ||
| Line 497: | Line 471: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">1. MiRNA is not degraded throughout the reaction process.</span> |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 506: | Line 478: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">2. The two fusion proteins of dCas9 and split-HRP fragments have the |
| − | + | ||
| − | + | ||
same ability to combine with the stem-loop structure, and only when two | same ability to combine with the stem-loop structure, and only when two | ||
| Line 521: | Line 491: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">3. The number of stem-loop structures in each RCA product is equal |
| − | + | ||
| − | + | ||
under a certain reaction time. </span> | under a certain reaction time. </span> | ||
| Line 532: | Line 500: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">4. The enzymatic activity remains unchanged with time under the premise |
| − | + | ||
| − | + | of excessive amount of enzymes or a short-time reaction.</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 543: | Line 510: | ||
<!--标题--> | <!--标题--> | ||
<h3> | <h3> | ||
| − | <span><span style="color:#7f1015"> About the data </span></span | + | <span><span style="color:#7f1015"> About the data </span></span> |
| − | + | ||
| − | + | ||
</h3> | </h3> | ||
| Line 551: | Line 516: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">1. The data we obtain from wet-lab experiment are reliable.</span> |
| − | + | ||
| − | reliable.</span> | + | |
</p> | </p> | ||
</br> | </br> | ||
| Line 560: | Line 523: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;"> | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">2. All the results are trustworthy in the process of statistical |
| − | + | processing and data calculation.</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 574: | Line 537: | ||
<!--标题--> | <!--标题--> | ||
<h3> | <h3> | ||
| − | <span><span style="color:#7f1015">Notations </span></span | + | <span><span style="color:#7f1015">Notations </span></span> |
</h3> | </h3> | ||
| Line 586: | Line 549: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>Symbol </span> | + | <span style="font-family:"">Symbol </span> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>Definition </span> | + | <span style="font-family:"">Definition </span> |
</p> | </p> | ||
</td> | </td> | ||
| Line 598: | Line 561: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span> | + | <i><span style="font-family:"">x</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK7"></a><a name="OLE_LINK6"></a><span>The concentration of</span><span> miRNA (pM)</span> | + | <a name="OLE_LINK7"></a><a name="OLE_LINK6"></a><span style="font-family:"">The |
| + | concentration of</span><span style="font-family:""> miRNA (pM)</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 610: | Line 574: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>C<sub>1</sub></span></i> | + | <i><span style="font-family:"">C<sub>1</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The concentration of initiated probe (Abbreviated | + | <span style="font-family:"">The |
| − | + | concentration of initiated probe (Abbreviated to iprobe)</span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 623: | Line 587: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>k<sub>1</sub></span></i> | + | <i><span style="font-family:"">k<sub>1</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK8"></a><span>A constant representing | + | <a name="OLE_LINK8"></a><span style="font-family:"">A constant representing the scale factor</span><span style="font-family:""></span> |
| − | + | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 636: | Line 599: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>K<sub>m </sub></span></i> | + | <i><span style="font-family:"">K<sub>m </sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>One of the characteristic constants of | + | <span style="font-family:"">One |
| − | + | of the characteristic constants of phi29 DNA polymerase</span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 649: | Line 612: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>V<sub>max</sub></span></i> | + | <i><span style="font-family:"">V<sub>max</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>One of the characteristic constants of phi29 | + | <span style="font-family:"">One |
| − | + | of the characteristic constants of phi29 DNA polymerase</span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 662: | Line 625: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>k<sub>2</sub></span></i> | + | <i><span style="font-family:"">k<sub>2</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>A constant representing the scale factor</span> | + | <span style="font-family:"">A |
| + | constant representing the scale factor</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 674: | Line 638: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>V</span></i> | + | <i><span style="font-family:"">V</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The initial speed of RCA </span> | + | <span style="font-family:"">The initial |
| + | speed of RCA </span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 686: | Line 651: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK9"></a><i><span>n<sub>1</sub></span></i><i><sub><span></span></sub></i> | + | <a name="OLE_LINK9"></a><i><span style="font-family:"">n<sub>1</sub></span></i><i><sub><span style="font-family:""></span></sub></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The moles of RCA product</span> | + | <span style="font-family:"">The |
| + | moles of RCA product</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 698: | Line 664: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>n<sub>2</sub></span></i> | + | <i><span style="font-family:"">n<sub>2</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK10"></a><span>The number of | + | <a name="OLE_LINK10"></a><span style="font-family:"">The number of stem-loop structures in each RCA product</span><span style="font-family:""></span> |
| − | + | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 711: | Line 676: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>n</span></i> | + | <i><span style="font-family:"">n</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The total amount of <a name="OLE_LINK23"></a><a name="OLE_LINK22"></a>stem-loop structures</span> | + | <span style="font-family:"">The total |
| + | amount of <a name="OLE_LINK23"></a><a name="OLE_LINK22"></a>stem-loop structures</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 723: | Line 689: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>N</span></i> | + | <i><span style="font-family:"">N</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK41"></a><a name="OLE_LINK16"></a><span>The molecule number of fusion | + | <a name="OLE_LINK41"></a><a name="OLE_LINK16"></a><span style="font-family:"">The |
| − | + | molecule number of the fusion protein of dCas9 and split-HRP fragments</span><span style="font-family:""></span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 736: | Line 702: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>k<sub>3</sub></span></i> | + | <i><span style="font-family:"">k<sub>3</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>A constant representing the scale factor</span> | + | <span style="font-family:"">A |
| + | constant representing the scale factor</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 748: | Line 715: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>y<sub>1</sub></span></i> | + | <i><span style="font-family:"">y<sub>1</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The fluorescence | + | <span style="font-family:"">The fluorescence |
| − | DNA-dye-complex (RFU)</span> | + | intensity of DNA-dye-complex (RFU)</span> |
</p> | </p> | ||
</td> | </td> | ||
| Line 761: | Line 728: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>N<sub>1</sub></span></i> | + | <i><span style="font-family:"">N<sub>1</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The molecule number of fusion protein of | + | <span style="font-family:"">The |
| − | + | molecule number of the fusion protein of dCas9 and split-HRP fragments in the | |
| + | solution</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 774: | Line 742: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>N<sub>2</sub></span></i> | + | <i><span style="font-family:"">N<sub>2</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK45"></a><span>The molecule number | + | <a name="OLE_LINK45"></a><span style="font-family:"">The molecule number of the fusion protein of dCas9 and |
| − | + | split-HRP fragments binding with stem-loop structure</span><span style="font-family:""></span> | |
| − | + | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 788: | Line 755: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>k<sub>4</sub></span></i> | + | <i><span style="font-family:"">k<sub>4</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>A constant representing the scale factor</span> | + | <span style="font-family:"">A |
| + | constant representing the scale factor</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 800: | Line 768: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span>k<sub>5</sub></span></i> | + | <i><span style="font-family:"">k<sub>5</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>A constant representing the scale factor</span> | + | <span style="font-family:"">A |
| + | constant representing the scale factor</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 812: | Line 781: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">ρ</span></i><i><span style="font-family:""></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>Signal to noise ratio(Abbreviated to | + | <span style="font-family:"">Signal |
| − | + | to noise ratio(Abbreviated to SNR)</span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 825: | Line 794: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">I</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The molecule number of formed intact HRP | + | <span style="font-family:"">The |
| − | + | molecule number of formed intact HRP proteins </span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 838: | Line 807: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">I<sub>1</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The molecule number of formed intact HRP | + | <span style="font-family:"">The |
| − | + | molecule number of formed intact HRP proteins in the solution</span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 851: | Line 820: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">I<sub>2</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <a name="OLE_LINK43"></a><span>The molecule number | + | <a name="OLE_LINK43"></a><span style="font-family:"">The molecule number of formed intact HRP proteins through |
| − | + | binding with stem-loop structure</span><span style="font-family:""></span> | |
</p> | </p> | ||
</td> | </td> | ||
| Line 864: | Line 833: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">t</span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>Reaction time</span> | + | <span style="font-family:"">Reaction |
| + | time</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 876: | Line 846: | ||
<td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="66" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <i><span style="font-family: | + | <i><span style="font-family:"">y<sub>2</sub></span></i> |
</p> | </p> | ||
</td> | </td> | ||
<td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | <td width="487" valign="top" style="border:solid windowtext 1.0pt;"> | ||
<p class="MsoNormal"> | <p class="MsoNormal"> | ||
| − | <span>The signal intensity (OD<sub>450</sub>)</span> | + | <span style="font-family:"">The |
| + | signal intensity (OD<sub>450</sub>)</span> | ||
</p> | </p> | ||
</td> | </td> | ||
| Line 888: | Line 859: | ||
</table> | </table> | ||
</p> | </p> | ||
| − | |||
| Line 894: | Line 864: | ||
<!--标题--> | <!--标题--> | ||
<h3> | <h3> | ||
| − | <span><span style="color:#7f1015"> The RCA model </span></span | + | <span><span style="color:#7f1015"> The RCA model </span></span> |
| − | + | ||
| − | + | ||
</h3> | </h3> | ||
| Line 903: | Line 871: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Firstly, we | |
| + | |||
| + | assume that there is a linear relationship between the | ||
concentration of the initiated probes and miRNA as the combination reaction | concentration of the initiated probes and miRNA as the combination reaction | ||
| Line 909: | Line 879: | ||
between miRNA and probe occurs spontaneously<sup>1 </sup> . This can be written | between miRNA and probe occurs spontaneously<sup>1 </sup> . This can be written | ||
| − | as: | + | as:</span> |
| − | + | ||
| − | </span> | + | |
</p> | </p> | ||
</br> | </br> | ||
| + | |||
<!--插入图片--> | <!--插入图片--> | ||
| Line 958: | Line 927: | ||
obvious that there is a linear relationship between the molecule numbers of | obvious that there is a linear relationship between the molecule numbers of | ||
| − | iprobe-dN (n<sub>1 </sub>, reflects the molecule numbers of RCA product) and the initial | + | iprobe-dN (n<sub>1 </sub> |
| + | , reflects the molecule numbers of RCA product) and the initial speed of RCA | ||
| − | + | reaction. Notably, under the premise of excessive amount of enzymes, the | |
| − | + | extending of each RCA production is related to the reaction time, not the | |
| − | + | concentration of the iprobe. It is a linear relationship when we assume that the | |
| − | + | enzymatic activity remains unchanged with time, thus, the number of stem-loop | |
| − | + | structures (n<sub>2 </sub> | |
| + | ) in each RCA product is stable under the premise of a certain reaction time. | ||
| − | + | </span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 982: | Line 953: | ||
</br> | </br> | ||
| − | + | <!--正文--> | |
| − | + | ||
| − | <!-- | + | |
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure 1. Schematic diagram</ | + | size:18px;"><b>Figure 1. Schematic diagram</b></span> |
</p> | </p> | ||
| − | |||
</br> | </br> | ||
| + | |||
| Line 1,025: | Line 994: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">And | <span style="line-height:2;font-family:Perpetua;font-size:18px;">And | ||
| − | then, SYBR Green binds to DNA. The resulting DNA-dye-complex absorbs blue light | + | then, SYBR Green binds to DNA. The |
| + | resulting DNA-dye-complex absorbs blue light (<i>λ<sub>max</sub></i> = 497nm) | ||
| − | + | and emits green light (<i>λ<sub>max</sub></i> = 520nm). Thus, there is | |
| + | a linear relationship between the total amount of stem-loop structures and the | ||
| − | + | fluorescence | |
| − | + | intensity of DNA-dye-complex. This can be written as:</span> | |
| − | + | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,057: | Line 1,027: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model6.jpg| | + | [[File:T--NUDT_CHINA--model6.jpg|210px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,067: | Line 1,037: | ||
<span><span style="color:#7f1015"> The signal detection model | <span><span style="color:#7f1015"> The signal detection model | ||
| − | </span></span | + | </span></span> |
</h3> | </h3> | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Firstly, it is | |
| − | + | obvious that there is a linear relationship between | |
the molecule number of formed intact HRP proteins in the solution (consider as | the molecule number of formed intact HRP proteins in the solution (consider as | ||
| − | NOISE) and the molecule number of fusion | + | NOISE) and the molecule number of the fusion proteins in the solution. This can |
| − | written as:</span> | + | be written as:</span> |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,099: | Line 1,069: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">Next, | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Next, | ||
| − | we | + | we build a probability model to find the expression of the molecule number of |
formed intact HRP proteins (fused with dCas9) through binding with stem-loop | formed intact HRP proteins (fused with dCas9) through binding with stem-loop | ||
| Line 1,107: | Line 1,077: | ||
structures and the molecule number of DNA-bound fusion proteins. We obtain the | structures and the molecule number of DNA-bound fusion proteins. We obtain the | ||
| − | result by Monte Carlo method. </span> | + | result by Monte Carlo method. </span> |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,122: | Line 1,092: | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure2. Frequency Distribution | + | size:18px;"><b>Figure2. Frequency Distribution</b> Plot of frequency |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | distribution of the molecule number of formed intact HRP proteins through | |
| + | |||
| + | binding with stem-loop structure. </span> | ||
| − | |||
| − | |||
</br> | </br> | ||
| Line 1,143: | Line 1,110: | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure 3. The result of the probability model | + | size:18px;"><b>Figure 3. The result of the probability model</b> Plot of the |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | expect value of the molecule number of formed intact proteins against the total | |
| − | + | amount of stem-loop structures, carried out at 0.1, 0.3, 0.5, 0.7, 0.9 of | |
| − | + | N<sub>2</sub> to n ratio.</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,171: | Line 1,135: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model8.jpg| | + | [[File:T--NUDT_CHINA--model8.jpg|70px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,179: | Line 1,143: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">As for | <span style="line-height:2;font-family:Perpetua;font-size:18px;">As for | ||
| − | + | <i>N<sub>2</sub></i>, | |
| − | + | which refers to the molecule number of the fusion proteins binding with | |
| − | loop structure. The equation is formulated based on the limiting case. The | + | stem-loop structure. The equation is formulated based on the limiting case. The |
| − | + | ||
equation can be written as:</span> | equation can be written as:</span> | ||
</p> | </p> | ||
| Line 1,192: | Line 1,155: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model9.jpg| | + | [[File:T--NUDT_CHINA--model9.jpg|105px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,201: | Line 1,164: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">And | <span style="line-height:2;font-family:Perpetua;font-size:18px;">And | ||
| − | then, we can obtain the expressions for ρ and I separately.</span> | + | then, we can obtain the expressions for <i>ρ</i></span> <span>and<i> I</i> |
| + | |||
| + | separately.</span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,210: | Line 1,175: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model10.jpg| | + | [[File:T--NUDT_CHINA--model10.jpg|50px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,223: | Line 1,188: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Notably, under | |
| − | + | the premise of excessive substrates, | |
| + | it is a linear relationship between the reaction rate and the concentration of | ||
| + | enzymes when we assume that the enzymatic activity remain unchanged with time, | ||
| + | thus, we can obtain the expression of the signal intensity at <i>t<sub>th</sub> | ||
| − | + | </i>time-step. This is | |
| − | + | written as:</span> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | as:</span> | + | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,242: | Line 1,205: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model12.jpg| | + | [[File:T--NUDT_CHINA--model12.jpg|70px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,252: | Line 1,215: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">In | <span style="line-height:2;font-family:Perpetua;font-size:18px;">In | ||
| − | summary, the relationship between the number of stem-loop structures and | + | summary, the relationship between the number of stem-loop structures and SNR |
| − | + | under the premise of adding a certain amount of the fusion proteins can be | |
| − | + | written as:</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,277: | Line 1,240: | ||
relationship between the number of Stem-loop structures and the signal intensity | relationship between the number of Stem-loop structures and the signal intensity | ||
| − | under the premise of adding a certain amount of fusion proteins | + | under the premise of adding a certain amount of the fusion proteins can be |
| − | as:</span> | + | written as:</span> |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,288: | Line 1,251: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model14.jpg| | + | [[File:T--NUDT_CHINA--model14.jpg|280px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,296: | Line 1,259: | ||
<span><span style="color:#7f1015"> The calculation of the constants | <span><span style="color:#7f1015"> The calculation of the constants | ||
| − | </span></span | + | </span></span> |
</h3> | </h3> | ||
| Line 1,303: | Line 1,266: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">To | <span style="line-height:2;font-family:Perpetua;font-size:18px;">To | ||
| − | simplify the equation (1), the constants | + | simplify the equation (1), the constants |
| − | + | can be integrated. As can be seen from the above table, <i>k<sub>1</sub></i>, | |
| − | from the above table, | + | |
| − | + | <i>k<sub>2</sub></i>, <i>k<sub>3</sub></i><sub> </sub>are | |
| + | constants representing the scale factor, <i>K<sub>m</sub></i> and | ||
| − | + | <i>V<sub>max</sub></i> are | |
| + | characteristic constants of phi 29 DNA polymerase, and <i>n<sub>2</sub></i>, | ||
| − | under the premise of a certain reaction time. Thus, define two constants, mark | + | which refers to the number of stem-loop structures |
| + | in each RCA product, is stable under the premise of a certain reaction time. | ||
| + | Thus, define two constants, mark as <i>a</i> and <i>b</i>, and then the equation | ||
| − | + | can be | |
| + | simplified as:</span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,320: | Line 1,287: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA--model15.jpg| | + | [[File:T--NUDT_CHINA--model15.jpg|105px|center]] |
<html> | <html> | ||
</br> | </br> | ||
| Line 1,326: | Line 1,293: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;">We | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">We use |
| − | this equation to fit the data points obtained through experiments. (Figure | + | this equation to fit the data points obtained through experiments. (Figure 2. |
| − | the | + | (D)in the <a href="/Team:NUDT_CHINA/Results">RESULTS.page</a>) The fitting |
| + | |||
| + | curve is shown below.</span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,342: | Line 1,311: | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure 4. Fitting curve | + | size:18px;"><b>Figure 4. Fitting curve</b> Fitting curve of fluorescence |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | intensity of DNA-dye-complex (RFU) against the concentrations of miRNA | |
| − | + | (pM).</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,363: | Line 1,329: | ||
follows: | follows: | ||
| − | + | <i>k<sub>3</sub> </i>=8.35*10<sup>-16 </sup> | |
| + | |||
| + | (<i>n</i>=<i>y<sub>1</sub></i>/<i>k<sub>3</sub></i>); <i>k<sub>4</sub></i> | ||
| + | |||
| + | =0.038;<i> k<sub>5</sub>t</i>=5.9*10<sup>-12</sup>. | ||
</span> | </span> | ||
</p> | </p> | ||
| Line 1,373: | Line 1,343: | ||
<span><span style="color:#7f1015"> Results and Analysis | <span><span style="color:#7f1015"> Results and Analysis | ||
| − | </span></span | + | </span></span> |
</h3> | </h3> | ||
| Line 1,380: | Line 1,350: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">By | <span style="line-height:2;font-family:Perpetua;font-size:18px;">By | ||
| − | integrating the two models, we can obtain the relationships between the | + | integrating the two models, we can obtain the relationships between SNR, the |
| − | + | signal intensity respectively and the concentration of miRNA under different | |
| − | + | addition amount of fusion proteins. | |
| + | </span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,403: | Line 1,374: | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | dimensional map of signal | + | size:18px;"><b>Figure |
| + | 5. The signal intensity (OD<sub>450</sub>)</b> Three-dimensional map of signal | ||
| − | additional amount of fusion proteins. </span> | + | intensity (OD<sub>450</sub>) |
| + | against miRNA concentration(pM) and additional amount of fusion proteins.</span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,422: | Line 1,390: | ||
exists a monotonous relation between the concentration of miRNA and the signal | exists a monotonous relation between the concentration of miRNA and the signal | ||
| − | intensity when the value of the molecule number of fusion proteins is | + | intensity when the value of the molecule number of the fusion proteins is |
| − | large. While the relationship does not hold when the value of the | + | relatively large. While the relationship does not hold when the value of the |
| − | number of fusion proteins is relatively small. And the signal | + | molecule number of the fusion proteins is relatively small. And the signal |
| − | increases as the value of the molecule number of fusion proteins | + | intensity increases as the value of the molecule number of the fusion proteins |
increases.</span> | increases.</span> | ||
| + | </p> | ||
| + | </br> | ||
| + | |||
| + | |||
| + | |||
| + | <!--图和图注--> | ||
| + | </br> | ||
| + | </html> | ||
| + | [[File:T--NUDT_CHINA--modelfig6.jpg|700px|center]] | ||
| + | <html> | ||
| + | </br> | ||
| + | <p> | ||
| + | <span style="line-height:2;font-family:Perpetua;font- | ||
| + | |||
| + | size:18px;"><b>Figure 6. The result of SNR</b> Three-dimensional map of signal | ||
| + | |||
| + | to noise ratio against miRNA concentration (pM) and additional amount of fusion | ||
| + | |||
| + | proteins.</span> | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,439: | Line 1,426: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">The | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The | ||
| − | + | signal-to-noise ratio decreases as the value of the molecule number of fusion | |
| − | + | proteins increases. </span> | |
| + | </p> | ||
| + | </br> | ||
| − | + | ||
| + | <!--标题--> | ||
| + | <h3> | ||
| + | <span><span style="color:#7f1015"> Test of model | ||
| + | |||
| + | </span></span> | ||
| + | </h3> | ||
| + | |||
| + | <!--正文--> | ||
| + | <p style="text-indent:22pt;"> | ||
| + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Our | ||
| + | |||
| + | model predicts the relationship between the signal intensity and the | ||
| + | |||
| + | concentration of miRNA under the premise of adding a certain amount of the | ||
| + | |||
| + | fusion proteins of dCas9 and split-HRP fragments. So we can test if our model is | ||
| + | |||
| + | reasonable by comparing the curves predicted by the model with those obtained | ||
| + | |||
| + | from the experiment. The results are shown below. | ||
| + | </span> | ||
</p> | </p> | ||
</br> | </br> | ||
| + | |||
| Line 1,451: | Line 1,462: | ||
</br> | </br> | ||
</html> | </html> | ||
| − | [[File:T--NUDT_CHINA-- | + | [[File:T--NUDT_CHINA--modelfig7-2.jpg|700px|center]] |
<html> | <html> | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure | + | size:18px;"><b>Figure 7. Test of model</b> Plot of the signal intensity (OD<sub>450</sub>) against miRNA concentration (pM) predicted by the model and experimented by wet-lab. (A) The value of the molecule number of each fusion protein added to the system is set to be 4.66e+13. The mass of each fusion protein added to the system in wet-lab is 0.5 ng. (B) The value of the molecule number of each fusion protein added to the system is set to be 4.66e+14. The mass of each fusion protein added to the system in wet-lab is 5 ng.</span> |
</p> | </p> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
</br> | </br> | ||
| + | |||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">We can learn | |
| − | + | from the figure above that the general trend of curves predicted by the model agree well with the experimental results. </span> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</br> | </br> | ||
| + | |||
| + | |||
| + | <!--标题--> | ||
| + | <h3> | ||
| + | <span><span style="color:#7f1015"> Model extension | ||
| + | |||
| + | </span></span> | ||
| + | </h3> | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">Furthermore, we | |
| − | + | ||
| − | size:18px;">Furthermore, we | + | |
| − | when the | + | consider that it is conducive to signal detection when the value of signal |
| − | than two. We obtain the ideal region through | + | intensity and SNR are both greater than two. We obtain the ideal region through |
| − | below. </span> | + | calculation, which is shown as below. </span> |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,503: | Line 1,513: | ||
</br> | </br> | ||
<p> | <p> | ||
| − | + | <span style="line-height:2;font-family:Perpetua;font- | |
| − | size:18px;">Figure | + | size:18px;"><b>Figure 8. Ideal region</b> Region of qualified logarithm of |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | concentrations of miRNA (pM) and logarithm of the additional amount of fusion | |
| − | + | proteins.</span> | |
</p> | </p> | ||
| + | |||
</br> | </br> | ||
| Line 1,522: | Line 1,530: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">When | <span style="line-height:2;font-family:Perpetua;font-size:18px;">When | ||
| − | the value of N is set to | + | the value of N is set to e+13.3, the range of the value of x contained in the |
| − | ideal region is | + | ideal region is a maximum, which means when the value of the molecule number of |
| − | + | the fusion proteins is e+13.3, the concentration range of miRNA that can be | |
| − | + | detected is maximum. </span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,549: | Line 1,557: | ||
<!--正文--> | <!--正文--> | ||
<p style="text-indent:22pt;"> | <p style="text-indent:22pt;"> | ||
| − | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The general trend of curves predicted by the model are in good agreement with the experimental |
| − | + | ||
| − | + | ||
| − | + | results, thus indicating that our model is reasonable.</span> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,563: | Line 1,567: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">The | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The | ||
| − | value of the molecule number of fusion protein of dCas9 and split-HRP fragments | + | value of the molecule number of the fusion protein of dCas9 and split-HRP |
| + | |||
| + | fragments not only affects the signal-to-noise ratio, but also the signal | ||
| − | + | intensity. So we need to weigh its impact on both to select the optimal | |
| − | + | solution.</span> | |
</p> | </p> | ||
</br> | </br> | ||
| Line 1,576: | Line 1,582: | ||
<span style="line-height:2;font-family:Perpetua;font-size:18px;">The | <span style="line-height:2;font-family:Perpetua;font-size:18px;">The | ||
| − | concentration range of miRNA | + | concentration range of miRNA that can be detected is a maximum when we set the |
| − | + | value of the molecule number of the fusion proteins to be e+13.3, building on | |
| − | + | which, our wet-lab protocol could be optimized in our future work.</span> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</br> | </br> | ||
| Line 1,593: | Line 1,597: | ||
<!--正文--> | <!--正文--> | ||
| − | <p | + | <p > |
| − | + | <span style="line-height:2;font-family:Perpetua;font-size:18px;">1 Deng, R. <i>et al</i>.Toehold-initiated rolling circleamplification for visualizing individual microRNAs in situ in single cells. <i>Angew Chem Int Ed Engl </i>53, 2389-2393,doi:10.1002/anie.201309388 (2014).</span> | |
| + | </p> | ||
| + | </br> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 1,618: | Line 1,635: | ||
</div> | </div> | ||
<!-- end #content-wrap --> | <!-- end #content-wrap --> | ||
| − | + | <div style="border:solid 1px rgb(10,31,84);background-color:rgb(10,31,84);"> | |
| − | + | <div class="row footer-link" style="border:solid 0px red;margin-top:100px;"> | |
| − | + | <style>.menu-link-container ul li{ list-style:none; } .menu-link-container a{ color:white; font-size:14px;line-height:26px; } | |
| − | + | .menu-link-container a:hover{ | |
| − | + | text-decoration:underline; | |
| − | + | } #c_menu9909 .medium-6{ text-align:center; width:auto; border:solid 0px red; } #c_menu9909 .medium-6 h5{color:white;}#c_menu9909 .medium-6{height:315px;border:solid 0px red;}</style> | |
| − | + | <div id="c_menu9909"> | |
| − | + | <!--div class="medium-6 columns" style="width:auto;"> | |
| − | + | <h5 class="column-header">HOME</h5> | |
| − | + | <div class="menu-link-container"></div></div--> | |
| − | + | <div class="medium-6 columns" style="width:auto;"> | |
| − | + | <h5 class="column-header">TEAM</h5> | |
| − | + | <div class="menu-link-container"> | |
| − | + | ||
| − | + | ||
| − | + | <a href="/Team:NUDT_CHINA/Team">Team</a><br/> | |
| − | + | ||
| − | + | <a href="/Team:NUDT_CHINA/Collaborations">Collaborations</a><br/> | |
| − | + | ||
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <h5 class="column-header">PROJECT</h5> | ||
| + | <div class="menu-link-container"> | ||
| + | |||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Description">Description</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Design">Design</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Experiments">Experiments</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Proof">Proof of Concept</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Demonstrate">Demonstrate</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Results">Results</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Notebook">Notebook</a><br/> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <h5 class="column-header">PARTS</h5> | ||
| + | <div class="menu-link-container"> | ||
| + | |||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Parts">Parts</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Basic_Part">Basic Parts</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Composite_Part">Composite Parts</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Part_Collection">Part Collection</a><br/> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <a href="/Team:NUDT_CHINA/Safety" style="text-decoration:none;"> | ||
| + | <h5 class="column-header">SAFETY</h5></a> | ||
| + | |||
| + | </div> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <a href="/Team:NUDT_CHINA/Attributions" style="text-decoration:none;"> | ||
| + | <h5 class="column-header">ATTRIBUTIONS</h5></a> | ||
| + | |||
| + | </div> | ||
| + | <!--div class="medium-6 columns" style="width:auto;"> | ||
| + | <A href="/Team:NUDT_CHINA/Attributions" style="text-decoration:none;"><h5 class="column-header">ATTRIBUTIONS</h5></A> | ||
| + | <div class="menu-link-container"></div></div--> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <h5 class="column-header">HUMAN | ||
| + | <br>PRACTICES</h5> | ||
| + | <div class="menu-link-container"> | ||
| + | |||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Human_Practices">Human Practices</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/HP/Silver">Silver</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/HP/Gold">Gold</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Integrated_Practices">Integrated Practices</a><br/> | ||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Engagement">Engagement</a><br/> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <div class="medium-6 columns" style="width:auto;"> | ||
| + | <h5 class="column-header">AWARDS</h5> | ||
| + | <div class="menu-link-container"> | ||
| + | |||
| + | |||
| + | <a href="/Team:NUDT_CHINA/Model">Model</a><br/> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <div class="medium-6 columns" style="width:0px;height:0px;"></div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
<footer id="footer"> | <footer id="footer"> | ||
<div class="row footer-link"> | <div class="row footer-link"> | ||
| Line 1,768: | Line 1,786: | ||
--> | --> | ||
| − | + | <!-- to top edit by inksci---> | |
| + | <style> | ||
| + | #to_top{ | ||
| + | position:fixed; | ||
| + | bottom:100px; | ||
| + | right:0px; | ||
| + | width:60px; | ||
| + | height:60px; | ||
| + | font-size:14px; | ||
| + | line-height:60px; | ||
| + | text-align:center; | ||
| + | background-color:black; | ||
| + | color:rgb(246,168,0); | ||
| + | cursor:pointer; | ||
| + | } | ||
| + | </style> | ||
| + | <div onclick="top_it()" id="to_top"> | ||
| + | TOP | ||
| + | </div> | ||
| + | <script type="text/javascript"> | ||
| + | var oTop = document.getElementById("to_top"); | ||
| + | oTop.style.display="none"; | ||
| + | var top_break=false; | ||
| + | /** | ||
| + | * 回到页面顶部 | ||
| + | * @param acceleration 加速度 | ||
| + | * @param time 时间间隔 (毫秒) | ||
| + | **/ | ||
| + | var save_scrolltop=100000000000000; | ||
| + | window.onscroll = function(){ | ||
| + | |||
| + | |||
| + | var scrolltop = document.documentElement.scrollTop || document.body.scrollTop; | ||
| + | if(scrolltop>300){ | ||
| + | oTop.style.display="block"; | ||
| + | }else{ | ||
| + | oTop.style.display="none"; | ||
| + | } | ||
| + | if(scrolltop>save_scrolltop) | ||
| + | { | ||
| + | top_break=true; | ||
| + | } | ||
| + | save_scrolltop=scrolltop; | ||
| + | } | ||
| + | function top_it(){ | ||
| + | top_break=false; | ||
| + | goTop();return false; | ||
| + | } | ||
| + | function goTop(acceleration, time) { | ||
| + | if(top_break){ | ||
| + | return; | ||
| + | } | ||
| + | oTop.style.display="none"; | ||
| + | acceleration = acceleration || 0.1; | ||
| + | time = time || 16; | ||
| + | |||
| + | var x1 = 0; | ||
| + | var y1 = 0; | ||
| + | var x2 = 0; | ||
| + | var y2 = 0; | ||
| + | var x3 = 0; | ||
| + | var y3 = 0; | ||
| + | |||
| + | if (document.documentElement) { | ||
| + | x1 = document.documentElement.scrollLeft || 0; | ||
| + | y1 = document.documentElement.scrollTop || 0; | ||
| + | } | ||
| + | if (document.body) { | ||
| + | x2 = document.body.scrollLeft || 0; | ||
| + | y2 = document.body.scrollTop || 0; | ||
| + | } | ||
| + | var x3 = window.scrollX || 0; | ||
| + | var y3 = window.scrollY || 0; | ||
| + | |||
| + | // 滚动条到页面顶部的水平距离 | ||
| + | var x = Math.max(x1, Math.max(x2, x3)); | ||
| + | // 滚动条到页面顶部的垂直距离 | ||
| + | var y = Math.max(y1, Math.max(y2, y3)); | ||
| + | |||
| + | // 滚动距离 = 目前距离 / 速度, 因为距离原来越小, 速度是大于 1 的数, 所以滚动距离会越来越小 | ||
| + | var speed = 1 + acceleration; | ||
| + | window.scrollTo(Math.floor(x / speed), Math.floor(y / speed)); | ||
| + | |||
| + | // 如果距离不为零, 继续调用迭代本函数 | ||
| + | if(x > 0 || y > 0) { | ||
| + | var invokeFunction = "goTop(" + acceleration + ", " + time + ")"; | ||
| + | window.setTimeout(invokeFunction, time); | ||
| + | } | ||
| + | } | ||
| + | </script> | ||
| + | <!-- to top edit by inksci---> | ||
</body> | </body> | ||
</html> | </html> | ||
Latest revision as of 13:34, 19 October 2016
Abstract
This model is created to evaluate the effectiveness of initial design, and offers guidelines on how the system can (or must) be improved. (You can go to PROJECT. page to see more.) The core idea is to simulate the process of producing the signal which can be detected, and draw a conclusion by obtaining the relationship between the signal intensity and the concentration of miRNA.
Introduction
We create mathematical models of two aspects of our project, a RCA model and a signal detection model.
The RCA model is based on the Michaelis-Menten equation. We can obtain the relationship between the concentration of miRNA and the number of stem-loop structures through theoretical calculation, and we use our experimental results to calculate the parameters we introduced previously.
In the signal detection model, we combine a probability model with the kinetic equation of enzymatic reaction, so we can obtain the relationship between the number of stem-loop structures and the signal intensity under the premise of adding a certain amount of the fusion proteins of dCas9 and split-HRP fragments.
By integrating the two models, we can theoretically predict the impacts of the molecule number of proteins on the signal to noise ratio and explain the trend of the signal intensity variation against the change of the concentration of miRNA in our wet-lab work.
Assumption and Justification
About model
1. MiRNA is not degraded throughout the reaction process.
2. The two fusion proteins of dCas9 and split-HRP fragments have the same ability to combine with the stem-loop structure, and only when two different proteins next to each other, can they have the ability to catalyze substrate and produce signal.
3. The number of stem-loop structures in each RCA product is equal under a certain reaction time.
4. The enzymatic activity remains unchanged with time under the premise of excessive amount of enzymes or a short-time reaction.
About the data
1. The data we obtain from wet-lab experiment are reliable.
2. All the results are trustworthy in the process of statistical processing and data calculation.
Model
Notations
The RCA model
Firstly, we assume that there is a linear relationship between the concentration of the initiated probes and miRNA as the combination reaction between miRNA and probe occurs spontaneously1 . This can be written as:
Next, RCA is an enzymatic reaction, the reaction equation is written as: iprobe+dNTP→ iprobe-dN+PPi, under the premise of excessive amount of enzymes and dNTPs, the relationship between the concentration of iprobe and the initial speed of RCA can be described by the Michaelis-Menten equation. This can be written as：
It is obvious that there is a linear relationship between the molecule numbers of iprobe-dN (n1 , reflects the molecule numbers of RCA product) and the initial speed of RCA reaction. Notably, under the premise of excessive amount of enzymes, the extending of each RCA production is related to the reaction time, not the concentration of the iprobe. It is a linear relationship when we assume that the enzymatic activity remains unchanged with time, thus, the number of stem-loop structures (n2 ) in each RCA product is stable under the premise of a certain reaction time.
Figure 1. Schematic diagram
This can be written as:
And then, SYBR Green binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 497nm) and emits green light (λmax = 520nm). Thus, there is a linear relationship between the total amount of stem-loop structures and the fluorescence intensity of DNA-dye-complex. This can be written as:
In summary, the relationship between the concentration of miRNA and the fluorescence intensity of DNA-dye-complex can be written as:
The signal detection model
Firstly, it is obvious that there is a linear relationship between the molecule number of formed intact HRP proteins in the solution (consider as NOISE) and the molecule number of the fusion proteins in the solution. This can be written as:
Next, we build a probability model to find the expression of the molecule number of formed intact HRP proteins (fused with dCas9) through binding with stem-loop structure (consider as SIGNAL). It is related to the total amount of stem-loop structures and the molecule number of DNA-bound fusion proteins. We obtain the result by Monte Carlo method.
Figure2. Frequency Distribution Plot of frequency distribution of the molecule number of formed intact HRP proteins through binding with stem-loop structure.
Figure 3. The result of the probability model Plot of the expect value of the molecule number of formed intact proteins against the total amount of stem-loop structures, carried out at 0.1, 0.3, 0.5, 0.7, 0.9 of N2 to n ratio.
The result can be written as:
As for N2, which refers to the molecule number of the fusion proteins binding with stem-loop structure. The equation is formulated based on the limiting case. The equation can be written as:
And then, we can obtain the expressions for ρ and I separately.
Notably, under the premise of excessive substrates, it is a linear relationship between the reaction rate and the concentration of enzymes when we assume that the enzymatic activity remain unchanged with time, thus, we can obtain the expression of the signal intensity at tth time-step. This is written as:
In summary, the relationship between the number of stem-loop structures and SNR under the premise of adding a certain amount of the fusion proteins can be written as:
The relationship between the number of Stem-loop structures and the signal intensity under the premise of adding a certain amount of the fusion proteins can be written as:
The calculation of the constants
To simplify the equation (1), the constants can be integrated. As can be seen from the above table, k1, k2, k3 are constants representing the scale factor, Km and Vmax are characteristic constants of phi 29 DNA polymerase, and n2, which refers to the number of stem-loop structures in each RCA product, is stable under the premise of a certain reaction time. Thus, define two constants, mark as a and b, and then the equation can be simplified as:
We use this equation to fit the data points obtained through experiments. (Figure 2. (D)in the RESULTS.page) The fitting curve is shown below.
Figure 4. Fitting curve Fitting curve of fluorescence intensity of DNA-dye-complex (RFU) against the concentrations of miRNA (pM).
With regard to the equation (2), we set the parameters based on the experiments as follows: k3 =8.35*10-16 (n=y1/k3); k4 =0.038; k5t=5.9*10-12.
Results and Analysis
By integrating the two models, we can obtain the relationships between SNR, the signal intensity respectively and the concentration of miRNA under different addition amount of fusion proteins.
The results are shown below.
Figure 5. The signal intensity (OD450) Three-dimensional map of signal intensity (OD450) against miRNA concentration(pM) and additional amount of fusion proteins.
There exists a monotonous relation between the concentration of miRNA and the signal intensity when the value of the molecule number of the fusion proteins is relatively large. While the relationship does not hold when the value of the molecule number of the fusion proteins is relatively small. And the signal intensity increases as the value of the molecule number of the fusion proteins increases.
Figure 6. The result of SNR Three-dimensional map of signal to noise ratio against miRNA concentration (pM) and additional amount of fusion proteins.
The signal-to-noise ratio decreases as the value of the molecule number of fusion proteins increases.
Test of model
Our model predicts the relationship between the signal intensity and the concentration of miRNA under the premise of adding a certain amount of the fusion proteins of dCas9 and split-HRP fragments. So we can test if our model is reasonable by comparing the curves predicted by the model with those obtained from the experiment. The results are shown below.
Figure 7. Test of model Plot of the signal intensity (OD450) against miRNA concentration (pM) predicted by the model and experimented by wet-lab. (A) The value of the molecule number of each fusion protein added to the system is set to be 4.66e+13. The mass of each fusion protein added to the system in wet-lab is 0.5 ng. (B) The value of the molecule number of each fusion protein added to the system is set to be 4.66e+14. The mass of each fusion protein added to the system in wet-lab is 5 ng.
We can learn from the figure above that the general trend of curves predicted by the model agree well with the experimental results.
Model extension
Furthermore, we consider that it is conducive to signal detection when the value of signal intensity and SNR are both greater than two. We obtain the ideal region through calculation, which is shown as below.
Figure 8. Ideal region Region of qualified logarithm of concentrations of miRNA (pM) and logarithm of the additional amount of fusion proteins.
When the value of N is set to e+13.3, the range of the value of x contained in the ideal region is a maximum, which means when the value of the molecule number of the fusion proteins is e+13.3, the concentration range of miRNA that can be detected is maximum.
Conclusion
Based on our simulation, we came to the conclusion that:
The general trend of curves predicted by the model are in good agreement with the experimental results, thus indicating that our model is reasonable.
The value of the molecule number of the fusion protein of dCas9 and split-HRP fragments not only affects the signal-to-noise ratio, but also the signal intensity. So we need to weigh its impact on both to select the optimal solution.
The concentration range of miRNA that can be detected is a maximum when we set the value of the molecule number of the fusion proteins to be e+13.3, building on which, our wet-lab protocol could be optimized in our future work.
Reference
1 Deng, R. et al.Toehold-initiated rolling circleamplification for visualizing individual microRNAs in situ in single cells. Angew Chem Int Ed Engl 53, 2389-2393,doi:10.1002/anie.201309388 (2014).
TOP
