Team:Edinburgh/Project/Non-antibiotic-Markers/DhlA

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Alternative selectable and counter-selectable markers:
<br /><br />
Haloalkane dehalogenase <i> (dhlA) </i>
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Background
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<i>DhlA</i> is haloalkane dehalogenase from <i>Xanthobacter autotrophicus GJ10</i> <a href="#bibliography" onclick="expand('works-cited');">(Keuning, Janssen, & Witholt, 1985)</a>. It converts 1,2-dichloroethane (DCE) into the more toxic 2-chloroethanol and is successfully used as a counterselectable marker in plants <a href="#bibliography" onclick="expand('works-cited');">(Naested, Fennema, Hao, Andersen, Janssen, & Mundy, 1999)</a>. This is why we decided to assess its suitability as a counter-selectable marker in bacteria.
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Cloning
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PCR amplification of BS-<i>dhlA</i> with these <a class="cursor-pointer" onclick="expand('primers');">primers</a> resulted in no product.
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<br /><i>Forward primer: atga gaattc gcggccgc t tctaga gaggc tctat gataa atgc<br />
Reverse primer: gact ctgcag cggccgc t actagt a tta t tattc tgtct cggca aagtg
</i><br />
<img id="fig14" src="https://static.igem.org/mediawiki/2012/0/0d/Markers-fig14.JPG"><br />
<i>Figure 1: DNA gel of PCR amplification of BS-<i>dhlA</i>  with <i>dhlA</i>  designed primer resulted in no bands.</i><br />
<a class="cursor-pointer" onclick="collapse('primers');">Close the primers.</a><br />
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Characterization
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Plates
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To test how suitable <i>dhlA</i>  is as counterselectable marker, BS-control  and BS-<i>dhlA</i>  strains were grown in the presence or absence of DCA. Both strains showed similar growth in both the presence or absence of DCA (Figure 1).<br />
<img id="fig15" src="https://static.igem.org/mediawiki/2012/e/e4/Markers-fig15.JPG"><br />
Figure 1.Comparison of growth of BS-contol and BS-<i>dhlA</i>  at 0 ul and 20 ul DCA. Both strains showed similar growth in the presence or absence of DCA.			</p>
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Liquid cultures 
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Liquid cultures with different DCA concentrations of both BS-control and BS-<i>dhlA</i>  were prepared. No distinct difference between the growths of the two strains was evident (Figure 2).<br />
<img id="fig16" src="https://static.igem.org/mediawiki/2012/f/f8/Markers-fig16.JPG"><br />
Figure 2. Comparison of growth of BS-contol and BS-<i>dhlA</i>  in liquid cultures with varying DCA concentrations. Both strains showed similar growth.
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Conclusion:
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We extensively characterized the dehalogenase enzyme on plates and in liquid cultures.<br /><br />
We determined that it is not suitable as a counterselectable marker.
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Further plans:
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To BioBrick it as it can be used in bioremediation (it can break down halogenated compounds).
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<br /><br />
<a href="https://2012.igem.org/Team:Edinburgh/Project/Non-antibiotic-Markers/Sucrose-Hydrolase"><span class="intense-emphasis">&lt;&lt;Prev</span></a><span style="color:white;">___</span><a href="https://2012.igem.org/Team:Edinburgh/Project/Non-antibiotic-Markers/Plac-RFP-SacB"><span class="intense-emphasis">Next&gt;&gt;</span></a>
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