Team:Nevada/ccdB

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<p>'''Our Method:'''<html><a href="http://2010.igem.org/wiki/images/0/05/CcdB_method.png"><img src="http://2010.igem.org/wiki/images/0/05/CcdB_method.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p>
<p>'''Our Method:'''<html><a href="http://2010.igem.org/wiki/images/0/05/CcdB_method.png"><img src="http://2010.igem.org/wiki/images/0/05/CcdB_method.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p>
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<p>ccdB was amplified using Polymerase Chain Reaction (PCR) with primers designed to contain the specific BioBrick prefix and suffix.  The amplified fragment was then TOPO-cloned into a PCR-Blunt II vector (Invitrogen).  The vector was then cut with EcoRI and PstI restriction enzymes and the ccdB fragment was transformed into the iGEM compatible vector pSB1C3.</p>
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Revision as of 01:30, 25 October 2010

Ccdb Header new.png


 

ccdB

Finished_final.png ccdB minimal gene Team:Nevada/registry submissions


The ccdB gene is a known topoisomerase II poison and will kill most commercially available E. coli cell lines. The ccdB gene can be used as a selectable marker by placing it in the multicloning region of a plasmid and propagating it in ccdB resistant cell lines (e.g. DB3.1 from New England Biolabs). During cloning, the ccdB gene can be switched out for your gene of interest and transformed into a cell line that is susceptible to the toxic effects of ccdB (e.g NEBβ cells from New England Biolabs). Colonies that grow on plates should contain the plasmid and your gene of interest. If the ccdB gene is still present in the plasmid, the plasmid will kill any colonies where it is still present. This is an improvement to the current ccdB cell death gene part (BBa_P1010) as it does not contain the inactive ccdA gene or an unknown stuffer region.

 

 

 

 

 

 


Our Method:

ccdB was amplified using Polymerase Chain Reaction (PCR) with primers designed to contain the specific BioBrick prefix and suffix. The amplified fragment was then TOPO-cloned into a PCR-Blunt II vector (Invitrogen). The vector was then cut with EcoRI and PstI restriction enzymes and the ccdB fragment was transformed into the iGEM compatible vector pSB1C3.

 

 

 


Our Results:

 

 

 

 

 

 


Vectors Used:

 

 

 

 

 

 






Nevada CABNR.jpg NV INBRE Logo.jpg UNR ASUN logo.jpg Promega logo.jpg Invitrogen logo.jpeg