Difference between revisions of "Team:Sherbrooke/Parts"

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Part Documentation

Every single parts are designed to adapt the recombineering technique onto the BIOBOT platform. The fiability of our parts should enable the full automization of the recombineering process, a first step in MAGE automatization. To do so, we have built kill switches and positive selection markers to help a robot picking only the right colonies through easy and visual reporters and selectable markers.

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 kill switch, 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 kill switch).

BBa_K1744001

This part is a positive selection system for recombineering. It contains a kanamycin resistance gene Aph(3’)-I (aminoglycoside 3’-phosphotransferase, truncated version) with its native promoter as well as amilCP, a deep blue chromoprotein. amilCP gene is under the control of a strong constitutive promoter with a strong RBS to make it more intense than previous versions. The cassette is designed to be a double positive selection marker for recombineering. Once amplified with primer bearing homology to the targeted region, the cassette can be integrated in the genome through lambda red recombination. Once integrated, the colonies should become resistant to kanamycin and become pale blue overtime. However, the blue color is actually faint and really slow to become apparent.

BBa_K1744002

This part is a fully autonomous arabinose kill switch with a kanamycin resistance gene (truncated version) and amilCP. The araC gene is driven by its native promoter. It controls the expression of vcrx028 (a toxin) through the promoter PBAD. This promoter is inducible with arabinose and can be repressed with glucose (final concentration of 1 and 5%w/v, respectively). vcrx028 is a toxin gene isolated from pVCR94, a conjugative plasmid discovered in the cholera outbreak of 1994 in a Rwanda refugee camp. Then there is amilCP, a chromoprotein isolated from Acropora millepora that serves as a selection marker. Finally, we have the kanamycin resistance gene which is Aph(3’)-I (aminoglycoside 3’-phosphotransferase) and originate from pOK12. It was selected because the coding sequence had a deletion, making the sequence smaller. This is of particular importance for our project because we use this part for recombineering, which is greatly impaired if the length of the cassette used is bigger than 1.5 kb. This part is designed to be used in a recombineering experiment to make a clean deletion where no scars such as FRT sites are left from the experiment. To do so, we first delete the targeted region with our cassette amplified with homology on both sides of the region to delete and then use a medium with glucose (to repress the toxin) and with kanamycin 50 µg/mL to select the recombinants. Then, using a fusion PCR of both sides of the deletion, the cassette is removed and we can counterselect with arabinose (which induce the toxin and kills cells that did not recombined).To minimize the cassette length and maximize the recombination frequencies we recommend using phosphothiolated primers, 80 bp homologies at least on both sides of the targeted region, only use the part of the cassette PBAD-vcrx028-P1U2-amilCP-KanR and do the recombineering in a cell containing a plasmidic araC expression. Since amilCP’s expression is not strong enough to really be visible in a genomic context, unlike plasmidic context, amilCP protein production may therefore serve as an easy plasmid detection system to eliminate plasmidic background that could occur during recombineering experiments. The system is designed so it is easy to select the good cells with the correct phenotype and speed up the screening process that is often the longest part of the experiment.

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