- 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 naïve or selected population via DNA microarray analysis. Important considerations for oligonucleotide design, DNA library construction, recombineering, strain characterization, and selection are discussed.