![]() We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. ![]() Successful constructs can easily be identified through blue-white screening. We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. However, generation of donor plasmids typically requires multiple cloning and screening steps. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination.
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