From Design to Practice: A Comprehensive Tutorial for the Rapid Multiplex Engineering of Escherichia coli Using Antibiotic Resistance Markers

using antibiotic markers. We leverage a group of 15 antibiotic resistance cassettes, most of which can be readily included when designing double-stranded DNA donors intended for recombineering and purchased from several vendors. Using these methods, 10-15 defined modifications to a single host strain can be achieved in less than three weeks, using two-day editing cycles. We discuss sequences and protocols as well as the optimal design of genetic modifications and the associated DNA. Key features • Enables 10-15 genomic modifications in less than 3 weeks. • Includes a comprehensive tutorial on designing donor DNA as well as choosing the appropriate antibiotic markers. • Includes DNA sequences that are amenable to commercial DNA synthesis.