Team:Westminster/Results

From 2012.igem.org

(Difference between revisions)
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}
}
body {
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div#header div#logo {
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ul#navigation-list > li {
float: left;
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ul#navigation-list > li > ul {
ul#navigation-list > li > ul {
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}
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ul#navigation-list > li > ul.submenu li {
ul#navigation-list > li > ul.submenu li {
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}
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div#home-main-image {
div#home-main-image {
text-align: center;
text-align: center;
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background-color: #410000;
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border-bottom:1px #000 solid;
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div#the-content {
 +
position:relative;
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top:-19px;
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}
div#home-main-image img {
div#home-main-image img {
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width: 885px;
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height: 300px;
height: 300px;
margin-bottom: 20px;
margin-bottom: 20px;
border-radius: 20px;
border-radius: 20px;
 +
*/
}
}
div.text-box {
div.text-box {
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padding: 15px 20px;
padding: 15px 20px;
border-radius: 10px;
border-radius: 10px;
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}
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overflow: auto;
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float: left;
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width: 235px;
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margin: 10px 0px 50px 20px;
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border-radius: 10px;
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div#footer-box {
div#footer-box {
border-radius: 0 0 5px 5px;
border-radius: 0 0 5px 5px;
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margin-top:50px;
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 +
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 +
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 +
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 +
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 +
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 +
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 +
#control-btn
 +
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 +
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line-height: 0px;
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background: url('http://2012.igem.org/wiki/images/d/d0/Close-btn.png')  #000 no-repeat  2px 2px;
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<script type="text/javascript">
<script type="text/javascript">
$('document').ready(function() {
$('document').ready(function() {
-
$('li.submenu-holder').hover(function() {
+
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$(this).children('ul.submenu').stop(true).fadeIn('slow');
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// menu
-
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+
$('li.submenu-holder').hover(function() {
-
$(this).children('ul.submenu').stop(true).fadeOut('slow');
+
$(this).children('ul.submenu').stop(true).fadeIn('fast');
 +
}, function() {
 +
$(this).children('ul.submenu').stop(true).fadeOut('fast');
 +
});
 +
 
 +
              // hide standard team basics
 +
              $('#top-section').hide();
 +
  $('#bodyContent p:first').addClass("annoying-gap");
 +
 
 +
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$('#control-btn').click( function(){
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$bar.slideUp('fast');
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{
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$bar = $("#top-section").clone();
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$($bar.find("#search-controls")).remove();
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$($bar.find("#p-logo")).remove();
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$("#wrapper").prepend($bar);
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$bar.css("position", "absolute");
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$bar.css("top", "0px");
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$bar.css("left", "0px");
 +
$bar.css("background", "none");
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$bar.css("background", "rgba(0,0,0,.5)");
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$bar.css("height", "20px");
 +
$bar.css("border", "none");
 +
$bar.css("z-index", "100");
 +
$bar.css("color", "#000");
 +
 +
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 +
$bar.slideDown('fast');
 +
 +
$('#control-btn').mouseover( function(){
 +
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 +
}
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</script>
-
 
</head>
</head>
<body>
<body>
-
 
<div id="wrapper">
<div id="wrapper">
<div id="header">
<div id="header">
-
<div id="logo">
+
  <div id="control-btn" class="btn-cog"></div>
-
<a href="http://2012.igem.org/Team:Westminster">
+
 
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<img src="http://2012.igem.org/wiki/images/1/1e/Westminster-logo.png" alt="iSTEM" title="Back to home!" />
+
-
</a>
+
-
</div><!-- #logo -->
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<div id="navigation">
<div id="navigation">
<ul id="navigation-list">
<ul id="navigation-list">
-
<li><a href="http://2012.igem.org/Team:Westminster" class="navlink">Home</a></li>
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<li><a href="http://2012.igem.org/Team:Westminster" class="navlink active">Home</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Team" class="navlink">Team</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Team" class="navlink">Team</a></li>
-
<li><a href="http://igem.org/Team.cgi?year=2012&team_name=Westminster"class="navlink">Team Profile</a></li>
 
<li class="submenu-holder">
<li class="submenu-holder">
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<a href="http://2012.igem.org/Team:Westminster/Project/Overview"class="navlink active">Project</a>
+
<a href="http://2012.igem.org/Team:Westminster/Overview"class="navlink">Project</a>
 +
 
<ul class="submenu">
<ul class="submenu">
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<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Overview"class="navlink active">Overview</a></li>
+
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Overview"class="navlink">Overview</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Problem"class="navlink">The Problem</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Problem"class="navlink">The Problem</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Experiments"class="navlink">Experiments</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Experiments"class="navlink">Experiments</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Protocols"class="navlink">Protocols</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Protocols"class="navlink">Protocols</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Results"class="navlink">Results</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Results"class="navlink">Results</a></li>
 +
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Safety"class="navlink">Safety</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Safety"class="navlink">Safety</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Journal"class="navlink">Journal</a></li>
<li class="submenu-button"><a href="http://2012.igem.org/Team:Westminster/Journal"class="navlink">Journal</a></li>
 +
                            </a></li>
 +
                 
</ul>
</ul>
</li>
</li>
<li><a href="http://2012.igem.org/Team:Westminster/Parts"class="navlink">Parts</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Parts"class="navlink">Parts</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Modeling"class="navlink">Modeling</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Modeling"class="navlink">Modeling</a></li>
-
<li><a href="http://2012.igem.org/Team:Westminster/Notebook"class="navlink">Notebook</a></li>
+
<li><a href="http://2012.igem.org/Team:Westminster/Outreach"class="navlink">Outreach</a></li>
-
<li><a href="http://2012.igem.org/Team:Westminster/Safety"class="navlink">Safety</a></li>
+
                    <li><a href="http://2012.igem.org/Team:Westminster/Judging"class="navlink">Judging Criteria</a></li>
 +
 
 +
<li><a href="http://2012.igem.org/Team:Westminster/Attributions"class="navlink">Attributions</a></li>
<li><a href="http://2012.igem.org/Team:Westminster/Attributions"class="navlink">Attributions</a></li>
</ul><!-- #navigation-list -->
</ul><!-- #navigation-list -->
</div><!-- #navigation -->
</div><!-- #navigation -->
 +
            <div id="logo">
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                    <a href="http://www.igem.org" title="iGEM homepage"><img src="http://igem.org/wiki/images/d/dc/Igem-small-white.png" alt="iGEM logo" width="55" height="44" /></a>
 +
<a href="http://2012.igem.org/Team:Westminster">
 +
<img src="http://2012.igem.org/wiki/images/a/ae/Logo-white.png" alt="iSTEM - intelligent synthetic tumour eliminating machine" width="172" height="66" title="iSTEM - intelligent synthetic tumour eliminating machine" />
 +
</a>
 +
</div><!-- #logo -->
</div><!-- #header -->
</div><!-- #header -->
<div id="the-content">
<div id="the-content">
-
<div id="home-main-image">
+
<!--div id="home-main-image">
<img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" alt="Welcome" title="Welcome to our home!" />
<img src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" alt="Welcome" title="Welcome to our home!" />
-
</div><!-- #home-main-image -->
+
</div--><!-- #home-main-image -->
<div id="home-main-text">
<div id="home-main-text">
-
<h2>Lorem Ipsum</h2>
+
<h1>WIKI Notebook</h1>
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
  <p><b>WETLAB WEEK 1</b></p>
-
<div class="two-columns">
+
  <p><span style='background:red;
-
<img class="two-columns-image" src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" width="440" />
+
'>Wednesday</span></p>
-
<img class="two-columns-image" src="http://2012.igem.org/wiki/images/4/4d/Westminster_team.png" width="440" />
+
  <p><span style='color:red'>AIM:</span> The primers for
-
<h2>Lorem Ipsum</h2>
+
    amplification of the ALDH promoter sequences have arrived. These primers have
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
    the <span class=SpellE>Biobrick</span> overhangs. Primers were diluted to 100mM
-
<p>Cancer recurrence is one of the fears that almost every patient undergoing chemotherapy develops. Recent findings suggest that only a small fraction of the tumor cells, called Cancer Stem Cells (CSC) are able to drive the growth of the tumor. CSC also show an increased drug resistance, and could remain unaffected after chemotherapy, eventually resulting in the formation of a new tumor.</p>
+
    and working concentrations of primers was made up at 10mM. The first set of
-
</div><!-- .two-columns -->
+
    primers to amplify the four ALDH isoforms was set up. </p>
 +
  <p>PCR was set up using <span class=SpellE>Pfu</span> polymerase. Template used was whole cells (mammalian). Cells were thawed, spun
 +
    down and the supernatant removed and re-suspended in 50uL DMSO. We attempted to
 +
    amplify the following promoter sequences: ALDH1A1, ALDH2, ALDH1A3, ALDH3A1 (1)
 +
    and <span class=GramE>ALDH3A1(</span>2). </p>
 +
  <p>The cycle conditions were as follows:</p>
 +
  <p>Initial denaturation at 98<sup>0</sup>C for 30 seconds then
 +
    30 cycles with 98<sup>0</sup>C for 10 seconds, 54<sup>0</sup>C for 30 seconds
 +
    (annealing) and 72<sup>0</sup>C for 30 seconds. Final extension was at 72<sup>0</sup>C
 +
    for 10 minutes and then PCR was held at 4<sup>0</sup>C. </p>
 +
  <p><span style='color:red'>RESULTS: </span>No bands were seen
 +
    on the gel. </p>
 +
  <p><span style='color:red'>ANALYSIS: </span>It is possible that
 +
    the DNA template (addition of whole cells) did not become available in the
 +
    reaction. Other cellular components during <span class=SpellE>lysis</span> of
 +
    the cells may also have had an impact on the efficiency of the PCR reaction. We
 +
    have located a protocol for crude extraction of mammalian DNA and will repeat
 +
    the PCR using extracted DNA. </p>
 +
  <p>The extraction protocol was performed. (<span class=GramE>see</span> protocols) Sample 1 was used as DNA template in the next PCR reaction. </p>
 +
  <p><span class=SpellE>Nanodrop</span> readings were as follows:</p>
 +
  <p>Sample 1: 42.1ng/<span style='font-family:"Times New Roman"'>µ</span>L
 +
    with purity ration 260/280 = 1.47</p>
 +
  <p>Sample 2: 50.4ng/<span style='font-family:"Times New Roman"'> µ</span>L with purity ration 260/280 = 1.30</p>
 +
  <p>Sample 3: 46.4ng/<span style='font-family:"Times New Roman"'> µ</span>L with purity ration 260/280 = 1.28</p>
 +
  <p><span style='background:red;'>Thursday</span></p>
 +
  <p><span style='color:red'>AIM:</span> Amplification of ALDH
 +
    promoters. A PCR reaction was carried out as before with the same reaction mix
 +
    and PCR conditions as Wednesday. <span class=GramE>amplify</span> ALDH1A1 and
 +
    ALDH2. </p>
 +
  <p><span style='color:red'>RESULTS: </span><span
 +
style='color:black;'>Amplification of ALDH1A1 was
 +
    successful using the ALDH1A1-Fw and ALDH-<span class=SpellE>Rv</span> primers.
 +
    The band size was as expected. ALDH 2 was also amplified using ALDH2-Fw and
 +
    ALDH2-Rv primers.&nbsp; </span></p>
 +
  <p><span lang=EN-US><img width=362 height=197
 +
src="http://2012.igem.org/wiki/images/e/ee/Result_002.png" ></span></p>
 +
  <p><span style='color:red'>ANALYSIS: </span><span
 +
style='color:black;'>We are pleased with developments thus
 +
    far. Difficulties in amplification of ALDH3A1 using our the <span class=GramE>ALDH3A1(</span>1)
 +
    and ALDH3A1(2) primers was anticipated. Initial analysis in selecting an
 +
    ALDH3A1 promoter region revealed a number of different options. Additionally,
 +
    it was difficult to find commercial sequences which matched the gene sequence
 +
    identified in NCBI. Subsequent to ordering the primers, the Eukaryotic Promoter
 +
    Database was discovered. Primers for the EPD experimentally designed ALDH3A1
 +
    promoter have been ordered and will be tested when they arrive.&nbsp; ALDH1A1 was initially designed to be
 +
    quite large and also includes an illegal site. An experimentally tested ALDH1A1
 +
    promoter has been identified on EPD. There is significant overlap between this
 +
    EPD promoter sequence and the one we had designed. A new forward EPD designed
 +
    primer was ordered which would give a smaller promoter and also eliminate the
 +
    illegal site. </span></p>
 +
  <p><span style='color:black;
 +
background:red;'>FRIDAY</span></p>
 +
  <p><span style='color:red'>&nbsp;</span>We have decided to try a touchdown
 +
    PCR reaction in order to try to amplify the other parts, particularly ALDH1A1.
 +
    Touchdown PCR is a faster but a more crude method of amplification in
 +
    comparison to gradient PCR. After setting up the first PCR, we realised we had
 +
    made a mistake (Touchdown 1). A second touchdown (Touchdown 2) was set up and
 +
    run soon after with the corrections. We managed to get bands for all four
 +
    promotes. These were cut from the gel, gel extracted, cut with EP (and ES for
 +
    ALDH2), ligated and transformed into competent E coli. </p>
 +
  <p>Touchdown 1 cycle conditions were as follows:</p>
 +
  <p>Initial denaturation at 98<sup>0</sup>C for 30 seconds then
 +
    10 cycles with 98<sup>0</sup>C for 10 seconds, 62<sup>0</sup>C for 30 seconds
 +
    (annealing) and 72<sup>0</sup>C for 1 minute 10 seconds, and annealing
 +
    temperature decreasing by 1<sup>0</sup>C for each cycle. Second amplification
 +
    cycle was 20 cycles of 98<sup>0</sup>C for 30 seconds, 52<sup>0</sup>C for 10
 +
    seconds (annealing) and 72<sup>0</sup>C for 1 minute 10 seconds. Final
 +
    extension was at 72<sup>0</sup>C for 10 minutes and then PCR was held at 4<sup>0</sup>C.
 +
    NOTE: In the second cycle (20 cycles) denaturation time at 98<sup>0</sup>C
 +
    should have been 10 seconds and annealing time should have been 30 seconds.
 +
    This correction was included in Touchdown 2. Additionally, DNA template
 +
    concentration was increased in Touchdown 2, by increasing the volume from 1<span
 +
style='font-family:"Times New Roman"'>µ</span>L per reaction to 5<span
 +
style='font-family:"Times New Roman"'> µ</span>L per reaction</p>
 +
  <p><span style='color:red'>RESULTS: </span>Spurious bands were
 +
    obtained in the touchdown 1, but ALDH1A1 and ALDH2 were amplified.&nbsp; Touchdown 2 gave good results for ALDH2.
 +
    For ALDH 1A3 and number of bands were seen. A faint band was observed at the
 +
    expected size and this is the band which was cut from the gel. ALDH1A1 from gel
 +
    1, ALDH2 from gel 2 and ALDH3A1 from gel 2 were also cut from the gel and gel
 +
    purified. Extracted DNA was run on a gel to verify the gel extraction process. &nbsp;Gel extracted DNA was digested with EP
 +
    and ALDH2 was restricted with ES. The backbone was restricted with EP and ES <span
 +
class=SpellE>aswell</span>. Restriction digests were saved till the next day. </p>
 +
  <p>GEL 1-Touchdown 1</p>
 +
  <p><span lang=EN-US><img width=453 height=237
 +
src="http://2012.igem.org/wiki/images/6/62/Result_004.png"/></span></p>
 +
  <p>Gel 2-Touchdown 2</p>
 +
  <p><span lang=EN-US><img width=453 height=237
 +
src="http://2012.igem.org/wiki/images/c/c0/Result_006.png" v:shapes="Picture_x0020_2"></span></p>
 +
  <p><b>WEEK 2</b></p>
 +
  <p><span style='color:red'>Monday</span></p>
 +
  <p>The linker primers and the EPD primers have arrived today. &nbsp;Touchdown PCR to amplify the promoter
 +
    parts was set up. The touchdown PCR was as before. The forward <span
 +
class=GramE>ALDH1A1(</span>EPD) primer was paired with the reverse primer
 +
    ALDH1A1-RV. The EPD designed primers for ALDH3A1 were tested. </p>
 +
  <p><span lang=EN-US><img width=400 height=224
 +
src="http://2012.igem.org/wiki/images/7/74/Result_008.png" /></span></p>
 +
  <p><span style='color:red'>RESULTS: </span>It worked a dream!
 +
    We have got lovely bands for <span class=GramE>ALDH1A1(</span>EPD), ALDH2 and
 +
    ALDH3A1 (EPD). However the band observed for ALDH1A3 was almost twice the
 +
    expected size. The size expected was around 900bp and the size seen on the gel
 +
    is around 1500bp. Parts cut from the gel were ALDH1A1 (EPD), ALDH3A1 (EPD) and
 +
    ALDH2 (<span class=SpellE>Wmin</span>). DNA was gel extracted and purified. DNA
 +
    was then run on a gel to verify the extraction process. An overnight digestion
 +
    was set up to create the <span class=SpellE>Biobrick</span> overhang parts. Only
 +
    the ALDH1A3 digest from previously was kept. The other digests were discarded
 +
    at this point. </p>
 +
  <p><span style='color:red'>Tuesday</span></p>
 +
  <p>Today the restricted parts were cloned to the pSB1C3
 +
    backbone and transformed to <i>E. coli. &nbsp;</i>Parts cloned were the ALDH1A1 (EPD),
 +
    ALDH2, ALDH1A3 and ALDH3A1. Transformed <i>E.
 +
    coli </i>was plated to chloramphenicol plates which were incubated overnight at
 +
    37<sup>0</sup>C. &nbsp;More plated and
 +
    broth were made up ready for use. The parts required from DTU Denmark and Serrano
 +
    are being arranged for delivery to us. </p>
 +
  <p>The following promoter parts have been mad ready. </p>
 +
  <p>ALDH1A1 &#8211; <span style='color:red'>BBa_K940000</span></p>
 +
  <p>ALDH2 &#8211; <span style='color:red'>BBa_K940001</span></p>
 +
  <p>ALDH 1A3 &#8211; <span style='color:red'>Bba_K940002</span></p>
 +
  <p>ALDH3A1 &#8211; <span style='color:red'>BBa_K940003</span></p>
 +
  <p><span lang=EN-US><img width=350 height=219
 +
src="http://2012.igem.org/wiki/images/a/a8/Result_010.png" v:shapes="Picture_x0020_6"></span></p>
 +
  <p><span style='color:red'>Wednesday</span></p>
 +
  <p>The linker primers were hydrated ready for use. We are still
 +
    waiting for the parts from DTU and Serrano. There have been a number of issues
 +
    with delivery but we are hoping to have them sorted and the parts delivered by
 +
    Friday. Colony PCR was done to confirm the inserts ligated yesterday. </p>
 +
  <p><span lang=EN-US><img width=329 height=343
 +
src="http://2012.igem.org/wiki/images/5/52/Result_012.png" v:shapes="Picture_x0020_1"></span></p>
 +
  <p><span style='color:red'>RESULTS: </span>Only some of the <span
 +
class=GramE>colony PCR have</span> worked. This may be due to suboptimal primer
 +
    annealing temperatures. Colony PCR was carried out as 10µL volumes an annealing
 +
    temperature was 58<sup>0</sup>C using the <span class=SpellE>Pfu</span> polymerase. All colonies were inoculated to LB broth and grown overnight at 37<sup>0</sup>C
 +
    in a shaking incubator. </p>
 +
  <p><span style='color:red'>Thursday</span></p>
 +
  <p>Two samples of each of the LB cultures were selected and
 +
    plasmid extracted using the <span class=SpellE>QIAprep</span> kit. PCR was then
 +
    done on the extracted plasmid in order to confirm DNA insert. This was done as
 +
    we had not managed to get good bands for <span class=GramE>all the</span> colony PCR’s. The PCR results were variable. Again, this may have been due to
 +
    annealing temperatures used in PCR. The samples were analysed on the <span
 +
class=SpellE>nanodrop</span>. <span class=SpellE>Nanodrop</span> results were
 +
    as follows:</p>
 +
  <p>But it has now been found that the concentrations we have
 +
    are too low to send for sequencing and still have enough for sending to iGEM
 +
    parts registry. An attempt to concentrate the samples was made by putting the
 +
    samples in a spin vacuum. Disaster!!! I have mixed up the lids! Colonies were
 +
    quickly re-picked and inoculated to LB for growth overnight. </p>
 +
  <p><span style='color:red'>Friday</span></p>
 +
  <p>The parts have arrived from DTU Denmark. PCR has been set up
 +
    using <span class=SpellE>Pfu</span> Turbo. DNA template used was as follows:</p>
 +
  <p>ALDH1A1 <span style='color:red'>(BBa_K940000) </span>extracted
 +
    plasmid</p>
 +
  <p><span class=SpellE><span class=GramE>mCherry</span></span> <span
 +
style='color:red'>(</span><span lang=EN style='color:red;'>BBa_K678067) </span><span lang=EN style='color:black;'>DTU</span></p>
 +
  <p><span lang=EN style='color:black;'>terminator </span><span
 +
style='color:red'>(</span><span
 +
lang=EN style='color:red;'>BBa_K678067) </span><span lang=EN
 +
style='color:black;
 +
'>DTU</span></p>
 +
  <p><span lang=EN style='color:black;'>hygromycin </span><span
 +
lang=EN style='color:red;'>(BBa_K678049) </span><span lang=EN
 +
style='color:black;
 +
'>DTU</span></p>
 +
  <p><span lang=EN style='color:black;'>ALDH2 </span><span
 +
style='color:red'>(BBa_K940000) </span><span style='color:black;'>extracted plasmid</span></p>
 +
  <p><span lang=EN style='color:black;'>Neomycin </span><span
 +
style='color:red'>(</span><span
 +
lang=EN style='color:red;'>BBa_K678067) </span><span lang=EN
 +
style='color:black;
 +
'>DTU</span></p>
 +
  <p><span lang=EN style='color:black;'>Biobrick
 +
    backbone </span><span lang=EN style='color:red;'>(pSB1C3)</span><span lang=EN
 +
style='color:black;
 +
'> unrestricted </span></p>
 +
  <p><span style='color:red'>RESULTS: </span>Unfortunately we did
 +
    not get any bands. Plasmid was visible on the gels which were run. This may
 +
    indicate that the plasmid concentration was too high for the PCR reaction. Plasmid
 +
    extraction was done using <span class=SpellE>QIAplasmid</span> prep kit. PCR
 +
    was done on the extracted plasmid, but again the PCR was not very successful.</p>
 +
  <p><span lang=EN-US><img width=439 height=163
 +
src="http://2012.igem.org/wiki/images/f/f5/Result_014.png" v:shapes="Picture_x0020_3"></span></p>
 +
  <p><span style='color:red'>NEXT STEPS: </span>Plasmid template was diluted 1/1000
 +
    and another PCR set up using the <span class=SpellE>Pfu</span>. <span
 +
class=SpellE>Hygromycin</span> is the only part which was amplified. Mg<sup>2<span
 +
class=GramE>+ <span style='vertical-align:baseline'>&nbsp;was</span></span></sup> included in the
 +
    reaction mix this time. DTU had reported success when they used Mg<sup>2<span
 +
class=GramE>+ <span style='vertical-align:baseline'>.</span></span></sup> We
 +
    were able to amplify <span class=SpellE>hygromycin</span>. </p>
 +
  <p><span lang=EN-US><img width=471 height=182
 +
src="http://2012.igem.org/wiki/images/6/69/Result_016.png" v:shapes="Picture_x0020_7"></span></p>
 +
  <p><b>WEEK 3</b></p>
 +
  <p><span style='color:red'>Monday</span></p>
 +
  <p>The plasmid previously extracted was analysed on the <span
 +
class=SpellE>nanodrop</span>. The samples were made ready for sequencing and
 +
    for sending to iGEM. </p>
 +
  <p><span class=SpellE>Nanodrop</span> results were as follows:</p>
 +
  <p><span class=GramE>ALDH1A1(</span>1): 64.5ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    2.13</p>
 +
  <p><span class=GramE>ALDH1A1(</span>2): 348.5ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280
 +
    =2.20</p>
 +
  <p><span class=GramE>ALDH2(</span>1): 59ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    1.08</p>
 +
  <p><span class=GramE>ALDH2(</span>2): 66.8ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    1.80</p>
 +
  <p><span class=GramE>ALDH1A3(</span>2): 166.9ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    2.17</p>
 +
  <p><span class=GramE>ADH1A3(</span>2): 44.5ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    2.20</p>
 +
  <p><span class=GramE>ADH1A3(</span>2): 174.2ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    2.20</p>
 +
  <p><span class=GramE>ADH1A3(</span>2): 201.4ng/<span
 +
style='font-family:"Times New Roman"'>µ</span>L with purity ration 260/280 =
 +
    2.22</p>
 +
  <p>PCR using <span class=SpellE>Taq</span> polymerase was set
 +
    up. We may be able to amplify the parts using this different polymerase.
 +
    Unfortunately such products could not be used for user cloning. <span
 +
class=SpellE>Taq</span> PCR was set up according to manufacturer protocol. PCR
 +
    was run overnight. </p>
 +
  <p><span style='color:red'>TUESDAY</span></p>
 +
  <p><span style='color:black;'>The overnight
 +
    PCR was analysed. </span></p>
 +
  <p><span style='color:black;'>Promoter
 +
    parts were sent for sequencing. The parts were also packaged and sent to iGEM
 +
    registry. </span></p>
 +
  <p>ALDH1A1 &#8211; <span style='color:red'>BBa_K940000</span></p>
 +
  <p>ALDH2 &#8211; <span style='color:red'>BBa_K940001</span></p>
 +
  <p>ALDH 1A3 &#8211; <span style='color:red'>Bba_K940002</span></p>
 +
  <p>ALDH3A1 &#8211; <span style='color:red'>BBa_K940003</span></p>
 +
  <p><span style='color:red'>WEDNESDAY</span></p>
 +
  <p>CONCLUSION:</p>
 +
  <p>Unfortunately we have not been able to achieve all that we set out
 +
    to do in the lab. In the two and a half weeks which we had in the lab we were
 +
    able to amplify the promoter regions which we had designed and sent them to
 +
    iGEM registry. The parts are currently being sequenced and the sequencing
 +
    information will be added to the registry of parts. Lab work may continue after
 +
    wiki freeze as it would be good to test the plug-n-play assembly. </p>
 +
 
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Revision as of 20:25, 26 September 2012

WIKI Notebook

WETLAB WEEK 1

Wednesday

AIM: The primers for amplification of the ALDH promoter sequences have arrived. These primers have the Biobrick overhangs. Primers were diluted to 100mM and working concentrations of primers was made up at 10mM. The first set of primers to amplify the four ALDH isoforms was set up.

PCR was set up using Pfu polymerase. Template used was whole cells (mammalian). Cells were thawed, spun down and the supernatant removed and re-suspended in 50uL DMSO. We attempted to amplify the following promoter sequences: ALDH1A1, ALDH2, ALDH1A3, ALDH3A1 (1) and ALDH3A1(2).

The cycle conditions were as follows:

Initial denaturation at 980C for 30 seconds then 30 cycles with 980C for 10 seconds, 540C for 30 seconds (annealing) and 720C for 30 seconds. Final extension was at 720C for 10 minutes and then PCR was held at 40C.

RESULTS: No bands were seen on the gel.

ANALYSIS: It is possible that the DNA template (addition of whole cells) did not become available in the reaction. Other cellular components during lysis of the cells may also have had an impact on the efficiency of the PCR reaction. We have located a protocol for crude extraction of mammalian DNA and will repeat the PCR using extracted DNA.

The extraction protocol was performed. (see protocols) Sample 1 was used as DNA template in the next PCR reaction.

Nanodrop readings were as follows:

Sample 1: 42.1ng/µL with purity ration 260/280 = 1.47

Sample 2: 50.4ng/ µL with purity ration 260/280 = 1.30

Sample 3: 46.4ng/ µL with purity ration 260/280 = 1.28

Thursday

AIM: Amplification of ALDH promoters. A PCR reaction was carried out as before with the same reaction mix and PCR conditions as Wednesday. amplify ALDH1A1 and ALDH2.

RESULTS: Amplification of ALDH1A1 was successful using the ALDH1A1-Fw and ALDH-Rv primers. The band size was as expected. ALDH 2 was also amplified using ALDH2-Fw and ALDH2-Rv primers. 

ANALYSIS: We are pleased with developments thus far. Difficulties in amplification of ALDH3A1 using our the ALDH3A1(1) and ALDH3A1(2) primers was anticipated. Initial analysis in selecting an ALDH3A1 promoter region revealed a number of different options. Additionally, it was difficult to find commercial sequences which matched the gene sequence identified in NCBI. Subsequent to ordering the primers, the Eukaryotic Promoter Database was discovered. Primers for the EPD experimentally designed ALDH3A1 promoter have been ordered and will be tested when they arrive.  ALDH1A1 was initially designed to be quite large and also includes an illegal site. An experimentally tested ALDH1A1 promoter has been identified on EPD. There is significant overlap between this EPD promoter sequence and the one we had designed. A new forward EPD designed primer was ordered which would give a smaller promoter and also eliminate the illegal site.

FRIDAY

 We have decided to try a touchdown PCR reaction in order to try to amplify the other parts, particularly ALDH1A1. Touchdown PCR is a faster but a more crude method of amplification in comparison to gradient PCR. After setting up the first PCR, we realised we had made a mistake (Touchdown 1). A second touchdown (Touchdown 2) was set up and run soon after with the corrections. We managed to get bands for all four promotes. These were cut from the gel, gel extracted, cut with EP (and ES for ALDH2), ligated and transformed into competent E coli.

Touchdown 1 cycle conditions were as follows:

Initial denaturation at 980C for 30 seconds then 10 cycles with 980C for 10 seconds, 620C for 30 seconds (annealing) and 720C for 1 minute 10 seconds, and annealing temperature decreasing by 10C for each cycle. Second amplification cycle was 20 cycles of 980C for 30 seconds, 520C for 10 seconds (annealing) and 720C for 1 minute 10 seconds. Final extension was at 720C for 10 minutes and then PCR was held at 40C. NOTE: In the second cycle (20 cycles) denaturation time at 980C should have been 10 seconds and annealing time should have been 30 seconds. This correction was included in Touchdown 2. Additionally, DNA template concentration was increased in Touchdown 2, by increasing the volume from 1µL per reaction to 5 µL per reaction

RESULTS: Spurious bands were obtained in the touchdown 1, but ALDH1A1 and ALDH2 were amplified.  Touchdown 2 gave good results for ALDH2. For ALDH 1A3 and number of bands were seen. A faint band was observed at the expected size and this is the band which was cut from the gel. ALDH1A1 from gel 1, ALDH2 from gel 2 and ALDH3A1 from gel 2 were also cut from the gel and gel purified. Extracted DNA was run on a gel to verify the gel extraction process.  Gel extracted DNA was digested with EP and ALDH2 was restricted with ES. The backbone was restricted with EP and ES aswell. Restriction digests were saved till the next day.

GEL 1-Touchdown 1

Gel 2-Touchdown 2

WEEK 2

Monday

The linker primers and the EPD primers have arrived today.  Touchdown PCR to amplify the promoter parts was set up. The touchdown PCR was as before. The forward ALDH1A1(EPD) primer was paired with the reverse primer ALDH1A1-RV. The EPD designed primers for ALDH3A1 were tested.

RESULTS: It worked a dream! We have got lovely bands for ALDH1A1(EPD), ALDH2 and ALDH3A1 (EPD). However the band observed for ALDH1A3 was almost twice the expected size. The size expected was around 900bp and the size seen on the gel is around 1500bp. Parts cut from the gel were ALDH1A1 (EPD), ALDH3A1 (EPD) and ALDH2 (Wmin). DNA was gel extracted and purified. DNA was then run on a gel to verify the extraction process. An overnight digestion was set up to create the Biobrick overhang parts. Only the ALDH1A3 digest from previously was kept. The other digests were discarded at this point.

Tuesday

Today the restricted parts were cloned to the pSB1C3 backbone and transformed to E. coli.  Parts cloned were the ALDH1A1 (EPD), ALDH2, ALDH1A3 and ALDH3A1. Transformed E. coli was plated to chloramphenicol plates which were incubated overnight at 370C.  More plated and broth were made up ready for use. The parts required from DTU Denmark and Serrano are being arranged for delivery to us.

The following promoter parts have been mad ready.

ALDH1A1 – BBa_K940000

ALDH2 – BBa_K940001

ALDH 1A3 – Bba_K940002

ALDH3A1 – BBa_K940003

Wednesday

The linker primers were hydrated ready for use. We are still waiting for the parts from DTU and Serrano. There have been a number of issues with delivery but we are hoping to have them sorted and the parts delivered by Friday. Colony PCR was done to confirm the inserts ligated yesterday.

RESULTS: Only some of the colony PCR have worked. This may be due to suboptimal primer annealing temperatures. Colony PCR was carried out as 10µL volumes an annealing temperature was 580C using the Pfu polymerase. All colonies were inoculated to LB broth and grown overnight at 370C in a shaking incubator.

Thursday

Two samples of each of the LB cultures were selected and plasmid extracted using the QIAprep kit. PCR was then done on the extracted plasmid in order to confirm DNA insert. This was done as we had not managed to get good bands for all the colony PCR’s. The PCR results were variable. Again, this may have been due to annealing temperatures used in PCR. The samples were analysed on the nanodrop. Nanodrop results were as follows:

But it has now been found that the concentrations we have are too low to send for sequencing and still have enough for sending to iGEM parts registry. An attempt to concentrate the samples was made by putting the samples in a spin vacuum. Disaster!!! I have mixed up the lids! Colonies were quickly re-picked and inoculated to LB for growth overnight.

Friday

The parts have arrived from DTU Denmark. PCR has been set up using Pfu Turbo. DNA template used was as follows:

ALDH1A1 (BBa_K940000) extracted plasmid

mCherry (BBa_K678067) DTU

terminator (BBa_K678067) DTU

hygromycin (BBa_K678049) DTU

ALDH2 (BBa_K940000) extracted plasmid

Neomycin (BBa_K678067) DTU

Biobrick backbone (pSB1C3) unrestricted

RESULTS: Unfortunately we did not get any bands. Plasmid was visible on the gels which were run. This may indicate that the plasmid concentration was too high for the PCR reaction. Plasmid extraction was done using QIAplasmid prep kit. PCR was done on the extracted plasmid, but again the PCR was not very successful.

NEXT STEPS: Plasmid template was diluted 1/1000 and another PCR set up using the Pfu. Hygromycin is the only part which was amplified. Mg2+  was included in the reaction mix this time. DTU had reported success when they used Mg2+ . We were able to amplify hygromycin.

WEEK 3

Monday

The plasmid previously extracted was analysed on the nanodrop. The samples were made ready for sequencing and for sending to iGEM.

Nanodrop results were as follows:

ALDH1A1(1): 64.5ng/µL with purity ration 260/280 = 2.13

ALDH1A1(2): 348.5ng/µL with purity ration 260/280 =2.20

ALDH2(1): 59ng/µL with purity ration 260/280 = 1.08

ALDH2(2): 66.8ng/µL with purity ration 260/280 = 1.80

ALDH1A3(2): 166.9ng/µL with purity ration 260/280 = 2.17

ADH1A3(2): 44.5ng/µL with purity ration 260/280 = 2.20

ADH1A3(2): 174.2ng/µL with purity ration 260/280 = 2.20

ADH1A3(2): 201.4ng/µL with purity ration 260/280 = 2.22

PCR using Taq polymerase was set up. We may be able to amplify the parts using this different polymerase. Unfortunately such products could not be used for user cloning. Taq PCR was set up according to manufacturer protocol. PCR was run overnight.

TUESDAY

The overnight PCR was analysed.

Promoter parts were sent for sequencing. The parts were also packaged and sent to iGEM registry.

ALDH1A1 – BBa_K940000

ALDH2 – BBa_K940001

ALDH 1A3 – Bba_K940002

ALDH3A1 – BBa_K940003

WEDNESDAY

CONCLUSION:

Unfortunately we have not been able to achieve all that we set out to do in the lab. In the two and a half weeks which we had in the lab we were able to amplify the promoter regions which we had designed and sent them to iGEM registry. The parts are currently being sequenced and the sequencing information will be added to the registry of parts. Lab work may continue after wiki freeze as it would be good to test the plug-n-play assembly.