Team:Edinburgh/Project/Non-antibiotic-Markers/Gene-replacement-strategy

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Alternative selectable and counter-selectable markers:
<br /><br />
Two-step protocol for gene replacement using a selection-counterselection cassette
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This protocol is useful if you want to replace a gene X with another gene Y. The example used here will detail the replacement of the <i> Citrobacter freundii </i> cephalosporinase gene with the limonene synthase gene, what makes our cells less antibiotic resistant and more citrus-scented (Figure 1). The idea was pioneered by Edinburgh's 2010 iGEM team - you can read more about their BRIDGE (BioBrick Recombination In Direct Genomic Editing) project 
<a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">here</a><br /><br />
<img style="width:70%;padding-left:15%;" src="https://static.igem.org/mediawiki/2012/4/48/EdiGEM_-_Graph2.png">
<br /><br />
Figure 1: Two-step gene replacement strategy, simplified schematic.
<br /><br />
<ol style="text-align:justify;">
<li>	The first step in the process is creating homology arms by PCR for the selection-counterselection cassette that are homologous to the regions flanking the cephalosporinase gene. The arm upstream of the cassette should have an EcoRI site at its 5’ end and the arm downstream of the cassette should have a PstI site at its 3’ end.<br /><br /></li>
<li>	 The upstream arm can then be digested with EcoRI and the downstream arm with PstI and these can then be ligated to the selection-counterselection cassette that was digested with both enzymes.<br /><br /></li>
<li>	A PCR using the outer primer pair should next be done to generate lots of a single linear product containing all three components (the upstream arm + cassette + downstream arm).<br /><br /></li>
<li>	Cells should first be transformed with a lambda red plasmid such as pSC101-gbaA (commercially available from GeneBridges http://www.genebridges.com/). This plasmid allows the cell to take up linear pieces of DNA without degrading them.<br /><br /></li>
<li>	They can then be transformed with this linear piece of DNA and hopefully some of them will undergo homologous recombination, cutting out the cephalosporinase gene and replacing it with the selection-counterselection cassette. After this step, the cells should be plated onto a kanamycin-containing medium to <i>select</i> for the cells that have taken up the cassette.<br /><br /></li>
<li>	The second step involves the replacement of this cassette with the limonene synthase gene – the cells that have grown on the kanamycin plate should be transformed with single stranded DNA containing the appropriate upstream arm + limonene synthase (or any other BioBrick) + downstream arm.  Again, in some of the cells homologous recombination will cause the excision of the cassette and insertion of the limonene synthase gene into the vacated region.<br /><br /></li>
<li>	This time, in order to select for the cells that have lost the cassette (<i> counter-select </i> for the cells that have the cassette), they should be plated out onto media that contain sucrose – cells that can grow on this medium (and are not red) have lost the levansucrase gene, and so the cassette, and are therefore good candidates for having had the cephalosporinase gene replaced by the limonene synthase gene.<br /><br /></li>
<li>	Finally, grow the cells at a non-permissive temperature to remove the lambda red plasmid and enjoy your lemon-scented cells!</li>
</ol>
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<br /><br />
<a href="https://2012.igem.org/Team:Edinburgh/Project/Non-antibiotic-Markers/Plac-RFP-SacB"><span class="intense-emphasis">&lt;&lt;Prev</span></a><span style="color:white;">__</span>6/6</span><span style="color:white;">__</span><span class="subtle-emphasis">Next&gt;&gt;</span>
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