Team:Uppsala University/Catsac

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<p>In our project we have extensively used recombineering onto the bacterial chromosome. To aid this work, we have built a improved Cat-SacB selection-counterselection casette, which provides easy and reliable recombineering, and the possibility of scarless gene deletion.</p>
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<p>In our project we have extensively used recombineering onto the bacterial chromosome. To aid this work, we have built an improved <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K864150">cat-SacB selection-counterselection cassette</a>, which provides easy and reliable recombineering, and the possibility of scarless gene deletion.</p>
<a href="http://partsregistry.org/wiki/images/5/56/CatsacB1.jpg">
<a href="http://partsregistry.org/wiki/images/5/56/CatsacB1.jpg">
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<img src="http://partsregistry.org/wiki/images/5/56/CatsacB1.jpg" width=400></a>
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<img src="http://partsregistry.org/wiki/images/5/56/CatsacB1.jpg" width=400></a><br>
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<i>Cat-SacB strain and wild-type on Cm plate and sucrose plate</i>
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Using λ-red recombineering you can make sure that your construct have inserted in the right place by first insert the cat-sacB cassette in the place you want your wanted insert and plate on chloramphenicol which selects for all bacteria containing the cassette. Then do another λ-red with your wanted insert and then slect on sucrose plate, only the bacteria which has lost the cassette have got the right construct on the right place.</p><p>
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When doing λ-red recombineering, you can make sure that your construct have inserted in the right place by plating on chloramphenicol which selects for all bacteria containing the cassette. Then, do another λ-red with your insert of interest that will replace the cat-sacB cassette and select on a sucrose plate, which will ensure that only the bacteria which has lost the cat-SacB cassette will survive.</p><p>
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To make sure that the cat-sacB construct work we plated DH5alpha cells containing the cat-sacB on chloramphenicol plates and on sucrose plates with a negative control who does not contain the cassette. On the chloramphenicol plates the bacteria survives and on sucrose plate they die.</p>
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To confirm that the cat-SacB construct works, we streaked MG1655 cells containing the cat-sacB integrated on the chromosome on LA plates with chloramphenicol (12 µg/mL) or sucrose (1 mM) together with a negative control (wild type MG1655) which does not contain the cassette. On the chloramphenicol plates the bacteria carrying the cat-sacB survives, while on the sucrose plate they die.</p>
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Latest revision as of 22:35, 26 October 2012

Team Uppsala University – iGEM 2012


Cat-SacB

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In our project we have extensively used recombineering onto the bacterial chromosome. To aid this work, we have built an improved cat-SacB selection-counterselection cassette, which provides easy and reliable recombineering, and the possibility of scarless gene deletion.


Cat-SacB strain and wild-type on Cm plate and sucrose plate

When doing λ-red recombineering, you can make sure that your construct have inserted in the right place by plating on chloramphenicol which selects for all bacteria containing the cassette. Then, do another λ-red with your insert of interest that will replace the cat-sacB cassette and select on a sucrose plate, which will ensure that only the bacteria which has lost the cat-SacB cassette will survive.

To confirm that the cat-SacB construct works, we streaked MG1655 cells containing the cat-sacB integrated on the chromosome on LA plates with chloramphenicol (12 µg/mL) or sucrose (1 mM) together with a negative control (wild type MG1655) which does not contain the cassette. On the chloramphenicol plates the bacteria carrying the cat-sacB survives, while on the sucrose plate they die.



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