Team:Edinburgh/Project/Citrobacter-Freundii/1-Replicon-compatibility

{{:Team:Edinburgh/header}}

<html>
<style type="text/css">
/* wiki edit */
#content{
	background-image:none;
}
/* header edit */
#project-page a{
	display:block;
	background-color: #f2f2f2;
	border-top-right-radius: 10px;
	border-top-left-radius: 10px;
}
#project-page a,
#project-page a:visited{
	color: #000;
}
#project-page a:hover{
	color: #002BB8;
}
/* actual page */
#page-content{
	background-color:#fff;
	border:1px solid #ccc;
}
#table02-method{
	height:0px;
	position: relative;
	top:-9000px;
}
#fig01,#table02{
	width:100%;
}
</style>
<script language="JavaScript">
	function expand(x){
		document.getElementById(x).style.height="auto";
		document.getElementById(x).style.top="0px";
	}
	function collapse(x){
		document.getElementById(x).style.height="0px";
		document.getElementById(x).style.top="-9000px";
	}
</script>
</html>

{{:Team:Edinburgh/Project/navigation}}

<html>
	<div id="page-content">
		<div class="text">
			<p class="h1">
<i>Citrobacter freundii</i> Characterisation:
<br /><br />
Replicon compatibility
			</p>
			<p class="normal-text">
To even consider a new chassis for synthetic biology (and especially iGEM), it should first of all be able to replicate the various types of plasmids that are used to insert genes/BioBricks into it. To test this, our <i>C. freundii</i> was transformed with several plasmids containing the most commonly used replicons. The transformations were done according to standard protocol written for <i> E. coli </i> and the transformants were plated out onto media as indicated in Table 1 and shown in Figure 1.<br /><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/Lemon-table01.JPG"><br />
<b>Table 1</b> - Replicon compatibility media and results<br /><br />
<img id="fig01" src="https://static.igem.org/mediawiki/2012/7/7c/Lemon-fig01.JPG"><br />
<b>Figure 1</b> - Plates containing <i>E. coli</i> or <i>Citrobacter freundii</i> transformed with plasmids that have different replicons. Note that the top row shows <i>Citrobacter freundii</i> cells and the bottom row shows <i>E. coli</i> cells, except for the first plates in each row, which are switched around, for colour comparison.
<br /><br />
<b>These results show that all but one of the plasmids have successfully been transformed into both <i>E. coli</i> and <i>C. freundii</i>, therefore these replicons are compatible with <i>C. freundii</i>.</b>
<br /><br />
<i>C. freundii</i> cells with the multi-host plasmid (pTG262) did not grow at all. The most probable reason is that the <i>cmlR</i> gene (which confers chloramphenicol resistance) in pTG262 is a Gram positive one (from <i>Lactobacillus</i>) which works much less well even in <i>E. coli</i> than the standard iGEM <i>cmlR</i> gene, so if <i>C. freundii</i> is a little more chloramphenicol-sensitive or expresses it a little worse, it would explain why no growth was seen on this plate.<br/>
<br />
			</p>
			<p class="h2" style="padding-left:50px;">
pTG262 characterisation
			</p>
			<p class="normal-text">
One reason why the <i>C. freundii</i> cell transformed with pTG262 did not grow may be that the <i>C. freundii</i> cells are less resistant to chloramphenicol than <i>E. coli</i>, so they were plated onto plates with varying concentrations of chloramphenicol (Table 2) to see whether they grow at all. pSB2K3 was used as a negative control, as it does not have chloramphenicol resistance.<br /><br />
<img id="table02" src="https://static.igem.org/mediawiki/2012/b/be/Lemon-table02.JPG"><br />
<b>Table 2</B> - Table indicating the amount of chloramphenicol added to each (20ml) plate.<br />
<a class="cursor-pointer" onclick="expand('table02-method')">Method</a>
			</p>
			<p class="normal-text" id="table02-method">
<br />
<i>The plates were incubated for two days and growth was observed on the 5 and 10 μl chloramphenicol plates.  In order to assess whether growth on these plates was due to the activity of the resistance gene on the plasmid or due to some innate resistance to chloramphenicol, 20 μl of C. freundii containing the plasmid or 20 μl untransformed C. freundii were plated out onto LB agar containing 5, 6, 7, 8, 9 or 10 μl chloramphenicol and incubated at 37&deg;C overnight.</i>
<a class="cursor-pointer" onclick="collapse('table02-method')">Close the method.</a>
<br /><br />
			</p>
			<p class="normal-text">
The <i>C. freundii</i> + pTG262 plasmid grew on all plates to some extent whereas the no plasmid control grew only below 8 &mu;l chloramphenicol and there were fewer colonies on the plates compared to the <i>C. freundii</i> + pTG262. This suggests that the plasmid works, but very weakly.
			</p>
			<p class="normal-text" style="text-align:center">
<br /><br />
<p class="h2">
Conclusions
			</p>
			<p class="normal-text">
<ul style="list-style-type:circle;padding:10px;">
	<li>We characterised the compatibility of <i>Citrobacter freundii</i> with the major replicon types used by iGEM<br /><br /></li>
	<li>We extensively characterized the pTG262 plasmid to figure out why it was not working well in <i>Citrobacter freundii</i><br /><br /></li>
	<li>We have concluded that the other replicons are compatible with this organism<br /><br /></li>

</ul>
			</p>
<p class="normal-text" style="text-align:center">
<br /><br />
<a href="https://2012.igem.org/Team:Edinburgh/Project/Citrobacter-Freundii/0-Introduction"><span class="intense-emphasis">&lt;&lt;Prev</span></a><span style="color:white;">__</span>2/7<span style="color:white;">__</span><a href="https://2012.igem.org/Team:Edinburgh/Project/Citrobacter-Freundii/2-Growth-in-sea-salts"><span class="intense-emphasis">Next&gt;&gt;</span></a>
<br /><br />
			</p>

		</div><!-- /text -->
	</div><!-- /page-content -->
</div><!-- /page-middle -->
</html>

<html>
<style type="text/css">
/* navigation edit*/
#page-navigation ul li#nav-lemon p,
#page-navigation ul li#nav-lemon ul li#nav-lemon-1 a{
	color:#ccc;
	cursor:default;
}
#page-navigation ul li#nav-lemon ul{
	height:auto;
}
#page-navigation ul li#nav-lemon ul li{
	top:0px;left:0px;
}
</style>
</html>