Difference between revisions of "Team:Kent/Results"

(Prototype team page)
 
 
(19 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
{{Kent}}
+
 
 +
 
 
<html>
 
<html>
 +
    <head>
 +
        <link href="https://maxcdn.bootstrapcdn.com/font-awesome/4.7.0/css/font-awesome.min.css" rel="stylesheet" integrity="sha384-wvfXpqpZZVQGK6TAh5PVlGOfQNHSoD2xbE+QkPxCAFlNEevoEH3Sl0sibVcOQVnN" crossorigin="anonymous">
 +
        <meta charset="utf-8">
 +
    </head>
 +
    <style>
 +
        .logo_2017 a img{
 +
        display:none;
 +
        }
 +
        #firstHeading{
 +
        display:none;
 +
        }
 +
        #sideMenu{
 +
        display: none;
 +
        }
 +
        @font-face {
 +
        font-family: 'KentExo2';
 +
        src: url(https://static.igem.org/mediawiki/2017/7/76/T--Kent--Exo2.ttf) format('truetype');
 +
        }
 +
        #globalWrapper, #content { /*-- changes default wiki settings --*/
 +
        margin: 0px;
 +
        padding: 0px;
 +
        width: 100%;
 +
        height: 100%;
 +
        border: 0px;
 +
        background: #272e33 url(https://static.igem.org/mediawiki/2017/a/ab/T--Kent--Resultsbkg.png) no-repeat center center fixed;
 +
        -webkit-background-size: cover;
 +
        -moz-background-size: cover;
 +
        -o-background-size: cover;
 +
        background-size: cover;
 +
        }
  
 +
        *{
 +
        padding: 0;
 +
        margin: 0;
 +
font-family: 'KentExo2';
 +
        }
  
−
<div class="column full_size" >
+
        #navdiv{
 +
        color: #FFFFFF;
 +
        left:0px;
 +
        padding: 30px;
 +
        text-align: center;
 +
        font-family:KentExo2;
 +
        position:fixed;
 +
        top:10px;
 +
        z-index:100;
 +
        }
  
−
<h1>Results</h1>
+
        #navdiv h1 img {
 +
        width:150px;
 +
        }
  
−
<p>Here you can describe the results of your project and your future plans. </p>
+
#navdiv h1 {
 +
line-height: 150px;
 +
margin-top:-25px;
 +
}
  
−
<h5>What should this page contain?</h5>
+
        #navdiv ul{
−
<ul>
+
background: transparent;
−
<li> Clearly and objectively describe the results of your work.</li>
+
        text-align: center;
−
<li> Future plans for the project. </li>
+
        list-style: none;
−
<li> Considerations for replicating the experiments. </li>
+
        padding: 0;
−
</ul>
+
        margin: 0;
 +
        display: block;
 +
                }
  
−
<h5>You should also describe what your results mean: </h5>
+
        #navdiv ul a{
 +
        text-decoration: none;
 +
        color: #FFFFFF;
  
−
<ul>
+
        }
−
<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
+
        #navdiv ul li{
−
<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
+
margin-top: 30px;
−
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
+
        float: left;
−
</ul>
+
        width: 169px;
 +
        height: 50px;
 +
        line-height: 50px;
 +
        position: relative;
 +
        text-transform: uppercase;
 +
        font-size: 16px;
 +
        color:#D0FEDC;
 +
        cursor: pointer;
 +
        opacity:0.8;
 +
border-radius: 10px;
 +
        }
  
−
</div>
+
#navdiv ul #teamLogo{
 +
margin-top:-10px;
 +
margin-left: 20px;
 +
margin-right: 20px;
 +
        float: left;
 +
        width: 150px;
 +
        height: 120px;
 +
        position: relative;
 +
        opacity:0.8;
 +
        }
  
−
<div class="clear"></div>
+
        #navdiv ul li:hover{
 +
border-radius:10px;
 +
        background: #28AAA1;
 +
        transition:all 0.3s;
 +
        opacity:0.9;
 +
        }
  
−
<div class="column half_size" >
 
  
 +
#navdiv ul #teamLogo:hover{
 +
border-radius:10px;
 +
        background: #455057;
 +
        transition:all 0.3s;
 +
        opacity:0.8;
 +
        }
  
−
<h5> Project Achievements </h5>
 
  
−
<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
+
        #navdiv ul li:hover a{
 +
        color:#455057;
 +
        }
  
−
<ul>
+
        #navdiv ul.drop-menu {
−
<li>A list of linked bullet points of the successful results during your project</li>
+
        position: absolute;
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
        top: 100%;
−
</ul>
+
        left: 0%;
 +
        width: 100%;
 +
        padding: 0;
 +
        z-index:100;
 +
        }
  
 +
        #navdiv ul.drop-menu li {
 +
        background: #455057;
 +
        color: #FFFFFF;
 +
       
 +
        }
 +
        #navdiv ul.drop-menu li:hover {
 +
color:#455057;
 +
        background: #35DBCB;
 +
 +
        }
 +
#navdiv ul.drop-menu li ul{
 +
        border-radius: 10px;
 +
        }
 +
     
 +
        #navdiv ul.drop-menu li {
 +
        display: none;
 +
        }
 +
 +
        #navdiv li:hover > ul.drop-menu{
 +
        perspective: 1000px;
 +
        }
 +
 +
        #navdiv li:hover > ul.drop-menu li {
 +
margin-top: 5px;
 +
        display: block;
 +
        text-align: center;
 +
        }
 +
 +
#title{
 +
margin-top:170px;
 +
}
 +
 +
        #title img{
 +
display:inline-block;
 +
        margin-left:12%;
 +
margin-right:12%;
 +
margin-bottom:100px;
 +
        width:230px;
 +
        }
 +
 +
#title span{
 +
position:absolute:
 +
margin-top:500px;
 +
display: inline-block;
 +
color:#28AAA1;
 +
font-size:50px;
 +
}
 +
 +
        .droptext {
 +
box-shadow: 0px 0px 0px 5px #28AAA1;
 +
border-radius:10px;
 +
        margin: auto;
 +
        margin-bottom:200px;
 +
        margin-top:50px;
 +
        width: 1200px;
 +
opacity:0.85;
 +
 +
}
 +
.droptext input {
 +
display: none;
 +
}
 +
.hull {
 +
position: relative;
 +
height: 100px;
 +
background: transparent;
 +
    transition: all .25s ease-in-out;
 +
}
 +
.hull::before {
 +
    content: '';
 +
    position: absolute;
 +
    display: block;
 +
    top: 0;
 +
    bottom: 0;
 +
    left: 0;
 +
    right: 0;
 +
    pointer-events: none;
 +
}
 +
header.hull {
 +
border-radius:10px 10px 0px 0px;
 +
border: 3px solid #455057;
 +
border-style:solid;
 +
border-bottom-color: #28AAA1;
 +
background: #455057;
 +
z-index: 50;
 +
cursor: initial;
 +
 +
}
 +
header .hull-title {
 +
margin: 0;
 +
font-weight: normal;
 +
font-size: 25pt;
 +
color: #28AAA1;
 +
cursor: initial;
 +
 +
}
 +
.hull-title {
 +
color:#def7f5;
 +
font-size: 20px;
 +
width: calc(100% - 40px);
 +
height: 50px;
 +
line-height: 50px;
 +
padding: 20px 0 0 50px;
 +
display: inline-block;
 +
cursor: pointer;
 +
-webkit-touch-callout: none;-webkit-user-select: none;-khtml-user-select: none;-moz-user-select: none;-ms-user-select: none;user-select: none;
 +
}
 +
.hull-content {
 +
width: calc(100% - 40px);
 +
padding: 30px 0 20px 20px;
 +
font-size: 11pt;
 +
color: #455057;
 +
display: none;
 +
}
 +
.hull-close {
 +
position: absolute;
 +
height: 64px;
 +
width: 100%;
 +
top: 0;
 +
left: 0;
 +
cursor: pointer;
 +
display: none;
 +
}
 +
input:checked + .hull {
 +
height: auto;
 +
color:#455057;
 +
background:#def7f5;
 +
border-top: 2.5px solid #28AAA1;
 +
 +
}
 +
input:checked + .hull .hull-title {
 +
color:#455057;
 +
}
 +
 +
input:checked + .hull .hull-content,
 +
input:checked + .hull .hull-close {
 +
display: inline-block;
 +
border-bottom: 2.5px solid #28AAA1;
 +
}
 +
 +
.arrows section .hull-title {
 +
padding-left: 44px;
 +
width: calc(100% - 80px);
 +
}
 +
input:checked + .arrows section .hull-title{
 +
color:#455057;
 +
}
 +
 +
.arrows section .hull-title:before {
 +
position: absolute;
 +
display: block;
 +
content: '\203a';
 +
font-size: 28pt;
 +
left: 20px;
 +
top: 17px;
 +
color: #28AAA1;
 +
transition: transform .5s ease-in-out;
 +
 +
}
 +
input:checked + section.hull .hull-title:before {
 +
transform: rotate(270deg);
 +
 +
}
 +
 +
#box1{
 +
margin:auto;
 +
width:1200px;
 +
text-align:center;
 +
}
 +
#textbox1{
 +
display:inline-block;
 +
margin: 40px 0 40px 0;
 +
width:1000px;
 +
border-radius:20px;
 +
opacity:0.8;
 +
background: #def7f5;
 +
border-style: solid;
 +
border-color: #28AAA1;
 +
text-align:center;
 +
}
 +
 +
#textbox2{
 +
display:inline-block;
 +
margin: 40px 0 40px 0;
 +
width:500px;
 +
border-radius:20px;
 +
opacity:0.8;
 +
background: #def7f5;
 +
border-style: solid;
 +
border-color: #28AAA1;
 +
text-align:center;}
 +
 +
 +
 +
#textbox3{
 +
display:inline-block;
 +
margin: 40px 0 40px 0;
 +
width:400px;
 +
border-radius:20px;
 +
opacity:0.8;
 +
background: #def7f5;
 +
border-style: solid;
 +
border-color: #28AAA1;
 +
text-align:center;}
 +
 +
#textbox4{
 +
display:inline-block;
 +
margin: 40px 0 40px 0;
 +
width:400px;
 +
border-radius:20px;
 +
opacity:0.8;
 +
background: #def7f5;
 +
border-style: solid;
 +
border-color: #28AAA1;
 +
text-align:center;}
 +
 +
#box1 p{
 +
display:inline-block;
 +
width: calc(100% - 40px);
 +
font-size:15px;
 +
color:#455057;
 +
}
 +
 +
 +
.centerizer{
 +
width:100%;
 +
text-align:center;
 +
margin: 50px 0 50px 0;
 +
}
 +
.centerizer span{
 +
color:#def7f5;
 +
font-size:50px;
 +
}
 +
 +
.lineSeparator{
 +
display:inline-block;
 +
width:850px;
 +
height:10px;
 +
background:#28AAA1;
 +
border-radius:10px;
 +
}
 +
.smallLineSeparator{
 +
display:inline-block;
 +
width:550px;
 +
height:10px;
 +
background:#28AAA1;
 +
border-radius:10px;
 +
}
 +
 +
#box1 img{
 +
width:500px;}
 +
        #foot ul{
 +
        display: inline-block;
 +
        position: fixed;
 +
        top:92%;
 +
        left:50%;
 +
        transform: translate(-50%,-50%);
 +
        }
 +
 +
        #foot ul{
 +
        display: flex;
 +
        position: fixed;
 +
        top:92%;
 +
        left:50%;
 +
        transform: translate(-50%,-50%);
 +
        }
 +
        #foot ul li{
 +
        list-style: none;
 +
        }
 +
        #foot ul li span{
 +
        width:50px;
 +
        height:50px;
 +
        background: #455057;
 +
        text-align:center;
 +
        line-height:50px;
 +
        font-size: 25px;
 +
        margin: 0 15px;
 +
        display: block;
 +
        border-radius: 50%;
 +
        position: relative;
 +
        overflow:hidden;
 +
        border: 4px solid #455057;
 +
        z-index:50;
 +
        }
 +
        #foot ul li span .fa{
 +
        margin:25%;
 +
        position:relative;
 +
        color:white;
 +
        transition: 0.5s;
 +
}
 +
 +
        #foot ul li span:hover .fa
 +
        {
 +
        color:#25343D;
 +
        transform: rotateY(360deg);
 +
        }
 +
        #foot ul li span:before
 +
        {
 +
        content:'';
 +
        position: absolute;
 +
        top:100%;
 +
        left:0;
 +
        width:100%;
 +
        height:100%;
 +
        background: #1AE195;
 +
        transition: 0.5s;
 +
        }
 +
        #foot ul li span:hover:before{
 +
        top:0;
 +
        }
 +
        #foot ul li:nth-child(1) span:before{
 +
        background: #E10C2B;
 +
        }
 +
        #foot ul li:nth-child(2) span:before{
 +
        background: #AA5EE7;
 +
        }
 +
        #foot ul li:nth-child(3) span:before{
 +
        background: #09F99E;
 +
        }
 +
        #foot ul li:nth-child(4) span:before{
 +
        background: #0367A6;
 +
        }
 +
        #foot ul li:nth-child(5) span:before{
 +
        background: #299CE4;
 +
        }
 +
        #foot ul li:nth-child(6) span:before{
 +
        background: #5BBBF7;
 +
        }
 +
 +
#sponsors{
 +
margin-left:50px;
 +
}
 +
#sponsors img{
 +
width:150px;
 +
}
 +
 +
#sponsorsdown #snapg img{
 +
margin-left:750px;
 +
}
 +
 +
    </style>
 +
 +
 +
 +
    <body>
 +
        <nav id="navdiv">
 +
            <ul>
 +
                <li>
 +
                    <a href="#">Project</a>
 +
                    <ul class="drop-menu menu-1">
 +
                        <a href="https://2017.igem.org/Team:Kent/Description"><li>Description</li></a>
 +
                      <a href="https://2017.igem.org/Team:Kent/Results"><li>Results</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Model"><li>Modelling</li></a>
 +
 +
                    </ul>
 +
                <li>
 +
                    <a href="#">Parts</a>
 +
                    <ul class="drop-menu menu-2">
 +
<a href="https://2017.igem.org/Team:Kent/Parts"> <li> Parts </li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Basic_Part"><li>Basic Parts</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Composite_Part"><li>Composite Parts</li></a>
 +
<a href = "https://2017.igem.org/Team:Kent/Part_Collection"><li> Part Collection </li></a>
 +
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Notebook</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Notebook"><li>Logbook</li></a>
 +
<a href="https://2017.igem.org/Team:Kent/Experiments"><li>Experiments</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Improve"><li>Future Ideas</li></a>
 +
                       
 +
                    </ul>
 +
                </li>
 +
                <li id="teamLogo">
 +
                    <a href="https://2017.igem.org/Team:Kent">
 +
                        <h1><img src="https://static.igem.org/mediawiki/2017/thumb/4/4f/T--Kent--Kenttemplogo.png/667px-T--Kent--Kenttemplogo.png"></h1>
 +
                    </a>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Safety</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Safety"><li>Project Safety</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Signs"><li>Hazard Signs</li></a>
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Team</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Team"><li>Meet the Team</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Contribution"><li>Contribution</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Attributions"><li>Attribution</li></a>
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Human Practices</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/HP/Silver"><li>Integrated Human Practices
 +
</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Engagement"><li>Public Engagement</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/InterLab"><li>Interlab</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Collaborations"><li>Collaboration</li></a>
 +
                    </ul>
 +
                </li>
 +
            </ul>
 +
        </nav>
 +
        <div id ="title">
 +
                <img src = "https://static.igem.org/mediawiki/2017/thumb/0/03/T--Kent--ResultsHeader.png/599px-T--Kent--ResultsHeader.png">
 +
<span>Results</span>
 +
<img src = "https://static.igem.org/mediawiki/2017/thumb/2/2f/T--Kent--ResultsHeader2.png/800px-T--Kent--ResultsHeader2.png">
 +
        </div>
 +
       
 +
<div id="box1">
 +
<div class="centerizer">
 +
 +
</div>
 +
<div id="textbox1" >
 +
<p>Our plan of creating our biobrick was pursued in two separate ways. First we attempted to clone the GFP with Standard 25 Prefix/Suffix from the biobrick library (BBa_K6480131) as well as the dCas13a we obtained from Addgene (L. buccalis C2C2, addgene #834852). We also ordered the whole gene through Integrated DNA Technologies (IDT)
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2017/f/f8/T--Kent--R1.png">
 +
<br>
 +
Figure 1. 1% agarose gel electrophoresis diagnostic restriction digestion results of GFP with Standard 25 Prefix/Suffix (BBa_K648013) in pSB1C3 . Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI; lane 4: single digestion with Pst1; lane 5: double digestion with EcoRI and PstI.
 +
</p>
 
</div>
 
</div>
 +
<div class="centerizer">
 +
<span>dCAS13a</span>
  
 +
<div class="lineSeparator"></div>
  
−
<div class="column half_size" >
+
<div id="textbox1" >
 +
<p><img src="https://static.igem.org/mediawiki/2017/1/1d/T--Kent--R2.png"><br>Figure 2:  1% agarose gel electrophoresis diagnostic restriction digestion results of L. buccalis C2C2, addgene #83485 in p2CT. Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI.
 +
<br>
 +
After successfully transforming these plasmids we designed primers for PCR amplification and Gibson Assembly3, adding a flexible linker sequence in between the GFP and the dCAS13a.
 +
<br>
 +
Because the dCAS13 gene contains three illegal restriction sites for iGEM submission (two EcoRI and one PstI) we also designed and ordered Quikchange4 PCR primers to remove these illegal sites.
 +
<br>
 +
After Gibson Assembly a screening PCR was performed with two screening primers, one 200 bp upstream and one 200bp downstream of the insert. Figure 3 shows a band the size approximately 4666 bp which indicates our insert (4266bp) plus an additional 400bp which account for the two screening primers on the 5’ and the 3’ end.
 +
<br>
 +
</p>
  
−
<h5>Inspiration</h5>
+
</div>
−
<p>See how other teams presented their results.</p>
+
−
<ul>
+
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
−
</ul>
+
  
 +
<div id="textbox1" >
 +
<p><img src="https://static.igem.org/mediawiki/2017/b/be/T--Kent--R3.png"><br>Figure 3. 1% agarose gel electrophoresis screening PCR results of dCAS13a - GFP in pSB1C3 . Lane 1: DNA Marker; Lane 2: negative control; lane 3; Screening PCR; lane 4: Screening PCR; lane 5: Screening PCR.
 +
<br>
 +
In order for our construct to work, it had to be transfected into HEK2935, a mammalian cell line. RNA localisation in bacteria is a contentious and poorly understood field. It does happen but not much is known about how, where and why6. In mammalian cells it is much better understood and poor localization has been linked with disease states7,8. Also because mammalian cells have cellular compartments, signals sequences can be added to the protein. We added nuclear localization sequences on both the N- and the C-terminal of the protein, so that unbound dCAS13a-GFP would localize in the nucleus until it is bound to an mRNA to track.
 +
<br>
 +
We transfected our construct (BBa_K2340000) with a guide RNA plasmid (BBa_K2340001 - BBa_K2340011) (1:2 ratio) into HEK293 cells using Lipofectamine™ 2000 in a 24 well plate in media and incubated for 24 hours at 37℃. This was viewed under a brightfield microscope at 100x magnification.
 +
<br>
 +
</p>
 
</div>
 
</div>
 +
<div id="textbox1" >
 +
<p><img src="https://static.igem.org/mediawiki/2017/8/81/T--Kent--R4.png"><br>Figure 4. Plate layout for transfection and brightfield microscopy. Well 1: Cas13 + Rab13 (1), well 2: Cas13 + Rab13 (2), well 3: Cas13 + Rab13 (3), well 4: Cas13 + EV, well 5: Rab13 (1) + EV, well 6: Pkp4 (1) + EV, well 7: Cas13 + Pkp4 (1), well 8: Cas13 + Pkp4 (2), well 9: Cas13 + Pkp4 (3), well 10: Inpp1 (1)+ EV, well 11: EV + ꞵ-actin (1), well 12: EV, well 13: Cas13 + Inpp1 (1), well 14: Cas13 + Inpp1 (2), well 15: Cas13 + Inpp1 (3), well 16: Transfection reagent control, well 17: parental, well 18: GFP control, well 19: Cas13 + ꞵ-actin (1), well 20: Cas13 + ꞵ-actin (2), well 21: Cas13 + ꞵ-actin (3). EV: Empty vector. Transfection reagent control: Lipofectamine™ 2000 GFP Control: eGFP in pcDNA3 (Addgene plasmid # 13031)
  
 +
</p>
 +
</div>
 +
<div id="textbox1" >
 +
<p>In Figure 5 we see the results from the brightfield microscopy. We saw no expression of our construct (Fig 5A) but the GFP control did express (Fig. 5B), indicating that there was transfection. The difference may lie in the type of GFP. This version of the construct had a wtGFP, which is natively expressed at lower temperatures (15-20 oC) and folds poorly at 37oC. We aim to redesign the GFP found on our construct to an eGFP (which is mutated to perform better at 37oC) to submit to the registry.   
 +
<br><img src="https://static.igem.org/mediawiki/2017/f/fb/T--Kent--R5.png"><br>Figure 5. Brightfield microscopy results. A: well 19: Cas13 + ꞵ-actin (1); no expression after 24 hours. After 30 and 48 hours there was still no expression (not shown here) B well 18: GFP Control; GFP expression after 24 hours.
  
  
 +
</p>
 +
</div>
 +
<span>Conclusion</span>
 +
 +
<div class="lineSeparator"></div>
 +
 +
<div id="textbox1" >
 +
<p>We successfully build one biobrick by fusing together a dCas13a gene with a wtGFP gene from the biobrick library, adding a flexible linker and two nuclear localization sequences to improve stability and reduce background noise. The function of our construct has not been able to proof due to the poor folding of the wtGFP at 37oC and the short time frame. We continued to tweak the design, mainly substituting the wtGFP to an eGFP. Future experiments could test how the protein would react with single a NLS and longer/shorter or rigid  linker sequences.
 +
</p>
 +
</div>
 +
<div class="centerizer">
 +
<span>References</span>
 +
 +
<div class="lineSeparator"></div>
 +
<div id="textbox1" >
 +
<ul><li>
 +
1Rose, J. (2011). GFP with Standard 25 Prefix/Suffix. [WWW Document]. URL http://parts.igem.org/Part:BBa_K648013
 +
</li><li>
 +
2Addgene (2016). p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A. [WWW Document]. URL http://www.addgene.org/83485/
 +
</li><li>
 +
3New England Biolabs (2017). Gibson Assembly® Master Mix. [WWW Document]. URL https://www.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
 +
</li><li>
 +
4Agilent genomics (2017). Quikchange. [WWW Document]. URL http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160
 +
</li><li>
 +
5Thomas P., Smart, T.G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51: 187-200.
 +
</li><li>
 +
6Avraam Buskila, A.., Kannaiah, S. & Amster-Choder, O. (2014). RNA localization in bacteria. RNA Biology, 11(8): 1051-1060.
 +
</li><li>
 +
7Pyke, C., et al. (1992). Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization. Cancer Research, 52: 1336-1341.
 +
</li><li>
 +
8Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D. (1998). RNA localization in development. Annu Rev Biochem, 67: 335-94.
 +
</li></ul>
 +
</div>
 +
</div>
 +
        <div id="foot">
 +
            <ul>
 +
                <li><span><a href="https://www.youtube.com/channel/UCV4kIcIodwiGjgEX_Rad1Ig"><i class="fa fa-youtube" aria-hidden="true"></i></a></span></li>
 +
                <li><span><a href="https://www.instagram.com/igem_kent2017/?hl=en"><i class="fa fa-instagram" aria-hidden="true"></i></a></span></li>
 +
                <li><span><a href="#"><i class="fa fa-envelope" aria-hidden="true"></i></a></span></li>
 +
                <li><span><a href="https://www.facebook.com/iGEMkent2017"><i class="fa fa-facebook-square" aria-hidden="true"></i></a></span></li>
 +
                <li><span><a href="https://www.linkedin.com/company/18159542/"><i class="fa fa-linkedin" aria-hidden="true"></i></a></span></li>
 +
                <li><span><a href="https://twitter.com/Kent_iGEM"><i class="fa fa-twitter" aria-hidden="true"></i></a></span></li>
 +
            </ul>
 +
        </div>
 +
<div id="sponsors">
 +
<div id="sponsorstop">
 +
<a href="https://www.kent.ac.uk/" id="ukc">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/a/a3/T--Kent--UKCwhite.png/800px-T--Kent--UKCwhite.png">
 +
</a>
 +
<a href="http://avokiddo.com/" id="avo">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/a/a7/T--Kent--avokiddo.png/800px-T--Kent--avokiddo.png">
 +
</a>
 +
</div>
 +
 +
<div id ="sponsorsdown">
 +
<a href="http://www.clicktravel.com" id="clickt">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/1/1e/T--Kent--clicktravel.png/799px-T--Kent--clicktravel.png">
 +
</a>
 +
<a href="http://www.bbsrc.ac.uk/" id="bbsrc">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/4/4e/T--Kent--bbsrc.png/800px-T--Kent--bbsrc.png">
 +
</a>
 +
<a href="http://www.snapgene.com/" id="snapg">
 +
<img src="https://static.igem.org/mediawiki/2017/3/36/T--Kent--snapgene.png">
 +
</a>
 +
</div>
 +
</div>
 +
    </body>
 
</html>
 
</html>

Latest revision as of 04:21, 16 December 2017


Results

Our plan of creating our biobrick was pursued in two separate ways. First we attempted to clone the GFP with Standard 25 Prefix/Suffix from the biobrick library (BBa_K6480131) as well as the dCas13a we obtained from Addgene (L. buccalis C2C2, addgene #834852). We also ordered the whole gene through Integrated DNA Technologies (IDT)

Figure 1. 1% agarose gel electrophoresis diagnostic restriction digestion results of GFP with Standard 25 Prefix/Suffix (BBa_K648013) in pSB1C3 . Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI; lane 4: single digestion with Pst1; lane 5: double digestion with EcoRI and PstI.

dCAS13a


Figure 2: 1% agarose gel electrophoresis diagnostic restriction digestion results of L. buccalis C2C2, addgene #83485 in p2CT. Lane 1: DNA Marker; Lane 2: undigested; lane 3; Single digestion with EcoRI.
After successfully transforming these plasmids we designed primers for PCR amplification and Gibson Assembly3, adding a flexible linker sequence in between the GFP and the dCAS13a.
Because the dCAS13 gene contains three illegal restriction sites for iGEM submission (two EcoRI and one PstI) we also designed and ordered Quikchange4 PCR primers to remove these illegal sites.
After Gibson Assembly a screening PCR was performed with two screening primers, one 200 bp upstream and one 200bp downstream of the insert. Figure 3 shows a band the size approximately 4666 bp which indicates our insert (4266bp) plus an additional 400bp which account for the two screening primers on the 5’ and the 3’ end.


Figure 3. 1% agarose gel electrophoresis screening PCR results of dCAS13a - GFP in pSB1C3 . Lane 1: DNA Marker; Lane 2: negative control; lane 3; Screening PCR; lane 4: Screening PCR; lane 5: Screening PCR.
In order for our construct to work, it had to be transfected into HEK2935, a mammalian cell line. RNA localisation in bacteria is a contentious and poorly understood field. It does happen but not much is known about how, where and why6. In mammalian cells it is much better understood and poor localization has been linked with disease states7,8. Also because mammalian cells have cellular compartments, signals sequences can be added to the protein. We added nuclear localization sequences on both the N- and the C-terminal of the protein, so that unbound dCAS13a-GFP would localize in the nucleus until it is bound to an mRNA to track.
We transfected our construct (BBa_K2340000) with a guide RNA plasmid (BBa_K2340001 - BBa_K2340011) (1:2 ratio) into HEK293 cells using Lipofectamine™ 2000 in a 24 well plate in media and incubated for 24 hours at 37℃. This was viewed under a brightfield microscope at 100x magnification.


Figure 4. Plate layout for transfection and brightfield microscopy. Well 1: Cas13 + Rab13 (1), well 2: Cas13 + Rab13 (2), well 3: Cas13 + Rab13 (3), well 4: Cas13 + EV, well 5: Rab13 (1) + EV, well 6: Pkp4 (1) + EV, well 7: Cas13 + Pkp4 (1), well 8: Cas13 + Pkp4 (2), well 9: Cas13 + Pkp4 (3), well 10: Inpp1 (1)+ EV, well 11: EV + ꞵ-actin (1), well 12: EV, well 13: Cas13 + Inpp1 (1), well 14: Cas13 + Inpp1 (2), well 15: Cas13 + Inpp1 (3), well 16: Transfection reagent control, well 17: parental, well 18: GFP control, well 19: Cas13 + ꞵ-actin (1), well 20: Cas13 + ꞵ-actin (2), well 21: Cas13 + ꞵ-actin (3). EV: Empty vector. Transfection reagent control: Lipofectamine™ 2000 GFP Control: eGFP in pcDNA3 (Addgene plasmid # 13031)

In Figure 5 we see the results from the brightfield microscopy. We saw no expression of our construct (Fig 5A) but the GFP control did express (Fig. 5B), indicating that there was transfection. The difference may lie in the type of GFP. This version of the construct had a wtGFP, which is natively expressed at lower temperatures (15-20 oC) and folds poorly at 37oC. We aim to redesign the GFP found on our construct to an eGFP (which is mutated to perform better at 37oC) to submit to the registry.

Figure 5. Brightfield microscopy results. A: well 19: Cas13 + ꞵ-actin (1); no expression after 24 hours. After 30 and 48 hours there was still no expression (not shown here) B well 18: GFP Control; GFP expression after 24 hours.

Conclusion

We successfully build one biobrick by fusing together a dCas13a gene with a wtGFP gene from the biobrick library, adding a flexible linker and two nuclear localization sequences to improve stability and reduce background noise. The function of our construct has not been able to proof due to the poor folding of the wtGFP at 37oC and the short time frame. We continued to tweak the design, mainly substituting the wtGFP to an eGFP. Future experiments could test how the protein would react with single a NLS and longer/shorter or rigid linker sequences.

References
  • 1Rose, J. (2011). GFP with Standard 25 Prefix/Suffix. [WWW Document]. URL http://parts.igem.org/Part:BBa_K648013
  • 2Addgene (2016). p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A. [WWW Document]. URL http://www.addgene.org/83485/
  • 3New England Biolabs (2017). Gibson Assembly® Master Mix. [WWW Document]. URL https://www.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
  • 4Agilent genomics (2017). Quikchange. [WWW Document]. URL http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160
  • 5Thomas P., Smart, T.G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51: 187-200.
  • 6Avraam Buskila, A.., Kannaiah, S. & Amster-Choder, O. (2014). RNA localization in bacteria. RNA Biology, 11(8): 1051-1060.
  • 7Pyke, C., et al. (1992). Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization. Cancer Research, 52: 1336-1341.
  • 8Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D. (1998). RNA localization in development. Annu Rev Biochem, 67: 335-94.