Trackable Multiplex Recombineering for Gene-Trait Mapping in E. coli

Recent advances in homologous recombination in Escherichia coli have enabled improved genome engineering by multiplex recombineering. In this chapter, we present trackable multiplex recombineering (TRMR), a method for gene-trait mapping which creates simulated knockdown and overexpression mutants for virtually all genes in the E . coli genome. The method combines oligonucleotide synthesis with multiplex recombineering to create two libraries comprising of over 8,000 E . coli strains in total that can be selected for traits of interest via high-throughput screening or selection. DNA barcodes included in the recombineering cassette allow for rapid characterization of a na�ve or selected population via DNA microarray analysis. Important considerations for oligonucleotide design, DNA library construction, recombineering, strain characterization, and selection are discussed.