Coselection of BAC for Escherichia coli chromosomal DNA multiplex automated genome engineering

Recombineering (recombination-mediated genetic engineering) is a powerful strategy for bacterial genomic DNA and plasmid DNA modifications. CoS-MAGE improved over MAGE (multiplex automated genome engineering) by co-electroporation of an antibiotic resistance repair oligo along with the oligos for modification of the Escherichia coli chromosome. After several cycles of recombineering, the sub-population of mutants were selected among the antibiotic resistant colonies. However, a pre-generated strain with mutS deletion and multiple inactivated antibiotic resistance genes integration is required. Herein, CoS-MAGE was modified by employing a single copy BAC vector harboring a bla-mkan cassette and a Red helper vector cloned with dominant mutL E32K , thus bypassing the utilization of the pre-generated strain. The proof-of-concept of the new strategy, CoS-BAC-MAGE, was demonstrated via the mutation of non-essential genes, essential genes, and AT rich regions of the wild type strain E. coli MG1655. With this system, an editing efficiency of 60% was realized. Furthermore, by toggling between two antibiotic resistance genes (one active, the other defective) on the BAC, sequential mutations were achieved without the requirement of BAC vector elimination and re-transformation. Via CoS-BAC-MAGE, simultaneously mutations of three sites were obtained in a day. We envision that CoS-BAC-MAGE will be a practical improvement for the generation of chromosomal mutations using the Cos-MAGE approach.