Two integration methods are widely used: single and double crossover. Before I chose which one to use, I want to make sure the advantages and disadvantages of them.
And by the way both of these phenomena may occur simultaneously within the host cell, and there is not much you can do to control the process. You may however, select for the single or double crossover clones, afterwards. Note that single crossovers resulting in double-stranded breaks would most likely cause cell death.
When you wanna do this in bacteria, there are two possibilitys. The plasmid you use can itegrate in your gene of interest by a singgle crossover. You interrupt the ORF by this integration. The best way to delete a gene is by double crossover, replacing the gene by an antibiotic cassette.
Thomas, I agree with you. Because single corssover just disrupt the target gene and might still leave partial function of the gene, and sometimes the insert is looped out. The double crossover can completely remove the whole gene.
A double crossover is better than single. In case of single crossover there is a chance of the multiple copies of the integrated genes, it might revert back to wild type. Double cross over are more stable, also the vector back bone can be removed and you can get only the desired gene integrated without the rest of the vector body.
Vectors are available to ensure that single and double integration occur in cells. There are markers on the plasmid which enable the selection
you can check out this paper , the vector is used for Lactococcous integration.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519346/
I hope it will help
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