Question

Explain how to make a successful plasmid construct that can knock out your intended target gene/homolog?

Answer 1

Answer: Plasmid is autonomously replicating extrachromosomal DNA. Natural plasmids are isolated from the bacterial cell and modified artificially to get the desired plasmid. The plasmid is used as a vector to transfer a gene of interest into the target cell. Plasmids vector are used to knock out specific genes in organisms.

A successful plasmid construct can be made that can knock out your intended target gene/homolog by integrating the gene sequence (knocked out) that has homology at the flank region. Homology in the flank region enables homologous recombination. Homologous recombination causes the wild-type gene to be replaced by the knocked-out gene integrated into the plasmid (also called recombineering). The plasmid has to be designed artificially. The plasmid should have an origin of replication for the formation of copies; an antibiotic resistance gene for selection; and a recognition site for gene integration. Each arm of the target gene should have a minimum of two kilobases (kb) pairs of nucleotides for the homologous recombination. However, for successful recombination six to 10 kb is desirable. PCR methods are used for generating copies of the plasmid vector. The gene sequence to be replaced with the original wild-type gene should have a marker gene for screening out successful recombinant cells.