High-throughput DNA engineering by mating bacteria

We introduce SCRIVENER (sequential conjugation and recombination for in vivo elongation of nucleotides with low errors), an in vivo DNA assembly platform that streamlines and scales DNA engineering. SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and homologous recombination to stitch DNA blocks together by mating E. coli in large arrays or pools. This approach is simpler, cheaper, and higher throughput than methods requiring DNA to be moved in and out of cells. We performed over 5,000 assemblies with 2 to 19 blocks (240 bp-12 kb) and assembled constructs up to 81 kb with high fidelity. Most errors are deletions between long repeats, but SCRIVENER minimizes their impact by enabling high-replication assembly and sequence verification at a nominal additional cost per replicate. The platform enables combinatorial library construction and DNA block reuse without PCR and is therefore a powerful tool to accelerate DNA design-build-test-learn cycles. A record of this paper's transparent peer review process is included in the supplemental information.