Difference between revisions of "Team:Alverno CA/Experiment"

 
(48 intermediate revisions by 9 users not shown)
Line 1: Line 1:
 
{{Alverno_CA}}
 
{{Alverno_CA}}
 
<html>
 
<html>
−
 
+
<script src="http://wayou.github.io/SlipHover/js/jquery.sliphover.min.js">
 +
    </script>
 +
    <script src="http://wayou.github.io/SlipHover/js/jquery.sliphover.min.js">
 +
    </script>
 +
    <style type="text/css">
 +
    #container{
 +
    width: 750px;
 +
    margin: 0 auto;
 +
                text-align:center;
 +
    }
 +
    #container img{
 +
    margin:10px 10px 0px 0px;
 +
    float: left;
 +
    }
 +
    #container div{
 +
    width: 650px;
 +
    overflow: hidden;
 +
    }
 +
    #container ul li{
 +
    list-style: none;
 +
    }
 +
        .sliphover-container{
 +
                z-index:99;
 +
        }
 +
    #menuContainer{
 +
    top:0px;
 +
    margin-top:0px;
 +
    position:absolute;
 +
    }
 +
    #top_nav_bulge{
 +
    top:110px;
 +
    }
 +
    </style>
 
<head>
 
<head>
−
<title>Alverno iGEM 2016</title>
 
−
<link rel="icon" href="https://static.igem.org/mediawiki/2016/thumb/5/58/T--Alverno_CA--Alverno_iGEM_2016_Logo.png/600px-T--Alverno_CA--Alverno_iGEM_2016_Logo.png">
 
−
</head>
 
−
 
 
<body>
 
<body>
−
<br>
 
−
<br>
 
−
<center><img src="https://static.igem.org/mediawiki/2016/thumb/5/58/T--Alverno_CA--Alverno_iGEM_2016_Logo.png/600px-T--Alverno_CA--Alverno_iGEM_2016_Logo.png" alt="Alverno iGEM Logo" style="width:300px;"></center>
 
−
<h2><center>Experiment</center></h2>
 
 
<br>
 
<br>
  
−
<h2>Introduction: </h2>
+
<center><h2>Experiment</h2></center>
  
−
<p> Our plasmids contain GFP coding device and RFP coding device, in varying orientations, with either a 500bp spacer, 1000bp spacer, or a dcas9 clamp binding site between them. They are on a vector with either Chloramphenicol or Kanamycin resistance. (For more details, see Design page —insert hyperlink!) After creating successful plasmids, we then transformed them in E. coli. The colonies were then inoculated and grown as overnight liquid cultures. </p>
+
<div class="demo" id="container">
 +
        <center>
 +
            <ul>
 +
                <li style="list-style: none"><br>
 +
                <br>
 +
                </li>
  
−
<h2>Experiment: </h2>
+
                <li><img src=
−
<h3> Plate Reader~ <h3>
+
                "https://static.igem.org/mediawiki/2016/5/52/T--Alverno_CA--Kiki_benchling.jpeg"
−
<h4> For in vivo using liquid cultures: </h4>
+
                style="height:215px;width:215px;" title=
−
<p> For cultures that contained colonies with plasmids that had a 500bp spacer or 1000bp spacer, we diluted these cultures to normalize them to the same OD absorbance. Then, we ran these in the plate reader to measure RFP (in AFU), GFP (in AFU), and OD (abs). For culture using a 1000bp spacer we tried testing the effects of using inducer as well. Using these results we were able to analyze the effects of supercoiling and the effects of using a base-pair spacer between them in order to reduce supercoiling. Because the GFP and RFP devices are placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (see Plate Reading protocol in Protocols: https://2016.igem.org/Team:Alverno_CA/Protocols) </p>
+
                "Using Benchling to design primers"/>
 +
                </li>
 +
                <li><img src=
 +
                "https://static.igem.org/mediawiki/2016/1/1e/Cultures_Experiment_page.jpeg"
 +
                style="height:215px;width:215px;" title=
 +
                "Liquid cultures of RFP/GFP constructs; transformed <i>E. coli</i>">
 +
                </li>
 +
                <li><img src=
 +
                "https://static.igem.org/mediawiki/2016/c/ca/T--Alverno_CA--Victoria_Plate_Reader2.jpg"
 +
                style="height:215px;width:215px;" title=
 +
                "Victoria using the plate reader">
 +
                </li>
 +
</center>
 +
</ul>
 +
       
 +
    </div>
 +
<br><br><br><br><br><br><br><br><br><br>
  
−
<h4> For in vitro using liquid plasmid DNA by TX-TL: </h4>
+
 
−
<p> For cultures that contained colonies that had a dcas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. We also designed and gRNA plasmids to use for testing as well and got dcas9 expression plasmids (DS-SPCasN- —insert hyperlink to AddGene!) from Caltech’s Murray Lab. All three of these plasmids were then expressed by in vitro using TX-TL. For our first run we did not use inducer, but for our second run we used ATc and IPTG to induce the TetR and LacI promoters on the GFP and RFP devices, respectively. After running this by TX-TL in the plate reader we obtained results that we then analyzed to determine whether or not supercoiling was reduced and GFP and RFP expressed. Once again, because the GFP and RFP devices are placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (see Plate Reading protocol in) </p>
+
<h3>Introduction </h3>
 +
 
 +
<h5> It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or <a href="https://www.addgene.org/66067/">DVK_AE</a> (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see <a href="https://2016.igem.org/Team:Alverno_CA/Design">Design</a>)
 +
 
 +
<br>After constructing these plasmids, we grew transformed <i>E. coli</i> as overnight liquid cultures. </h5>
 +
<br><br>
 +
<h4>Methods </h4>
 +
<br>
 +
<h3>Testing spacers <i>in vivo</i></h3>
 +
<h5>Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a <a href="http://www.perkinelmer.com/product/victor-x5-for-fl-lum-uv-trf-fp-2030-0050">VICTOR-X3</a> plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information) </h5>
 +
 
 +
<h3> Testing dCas9 clamps <i>in vitro</i> </h3>
 +
<h5> Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (<a href="https://www.addgene.org/48657/">DS-SPCasN</a>). All plasmids were mini-prepped and expressed in TX-TL <a href="http://www.openwetware.org/wiki/Biomolecular_Breadboards">TX-TL</a>, an <i>in vitro</i> prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information)</h5>
 +
 
 +
 
 +
<br>
 +
<h4><a href="https://2016.igem.org/Team:Alverno_CA/Protocols"> Click Here To Access More Protocols</a></h4>
  
  
−
<h4><a href="https://2016.igem.org/Team:Alverno_CA/Protocols">For More Protocols</a></h4>
+
<script>
 +
        $("#container").sliphover();
 +
        $(".sliphover-container").css('z-index','99');
 +
        $("#menuContainer").css('z-index','99');
 +
        </script>

Latest revision as of 20:02, 2 November 2016


Experiment













Introduction

It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or DVK_AE (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see Design)
After constructing these plasmids, we grew transformed E. coli as overnight liquid cultures.


Methods


Testing spacers in vivo

Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a VICTOR-X3 plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See "Plate Reading" for more information)

Testing dCas9 clamps in vitro

Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (DS-SPCasN). All plasmids were mini-prepped and expressed in TX-TL TX-TL, an in vitro prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See "Plate Reading" for more information)

Click Here To Access More Protocols