Difference between revisions of "Team:Sherbrooke/Parts"
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| − | + | An overview of the parts' design is shown in the picture below. | |
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| + | <h3>BBa_K1744000</h3> | ||
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| + | <a href="http://parts.igem.org/Part:BBa_K1744000">This part</a> is designed for clean deletion recombineering experiments. It has an arabinose induced toxin for negative selection and an ampicillin resistance gene for positive selection. This part is designed to be amplified using primer with homology to the targeted region and then integrated in that region using lambda red recombineering systems available commercially such as the heat-inducible pSIM plasmid family. The toxin vcrx028 is toxic for the cell. To repress its expression you must put glucose in the medium (we use 5%w/v). To activate the arabinose killswitch, we use 1 %w/v arabinose in the medium. To do the clean deletion (without DNA scar in the sequence) through recombineering, you must first insert the cassette in the genome by recombination, causing the deletion, and select the recombinants with ampicillin without forgetting to use a medium containing glucose to avoid unwanted toxin expression. Then you have to make the cassette pop out by recombineering using a fusion PCR of both adjacent regions to the one you want to delete and counterselect the cells that did not lose the part with arabinose (triggering the killswitch). | ||
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Revision as of 01:11, 16 September 2015
Part Documentation
An overview of the parts' design is shown in the picture below.
BBa_K1744000
This part is designed for clean deletion recombineering experiments. It has an arabinose induced toxin for negative selection and an ampicillin resistance gene for positive selection. This part is designed to be amplified using primer with homology to the targeted region and then integrated in that region using lambda red recombineering systems available commercially such as the heat-inducible pSIM plasmid family. The toxin vcrx028 is toxic for the cell. To repress its expression you must put glucose in the medium (we use 5%w/v). To activate the arabinose killswitch, we use 1 %w/v arabinose in the medium. To do the clean deletion (without DNA scar in the sequence) through recombineering, you must first insert the cassette in the genome by recombination, causing the deletion, and select the recombinants with ampicillin without forgetting to use a medium containing glucose to avoid unwanted toxin expression. Then you have to make the cassette pop out by recombineering using a fusion PCR of both adjacent regions to the one you want to delete and counterselect the cells that did not lose the part with arabinose (triggering the killswitch).
Overview of the design