Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function(1–3). To build large genomes rapidly, scalably, and in parallel we need: (1) methods for assembling megabases of DNA from shorter pre-cursors; and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS) - a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also develop continuous genome synthesis (CGS) - a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers(1,4) between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome – a key intermediate in its total synthesis(1) – from five episomes in 10 days. By parallelizing CGS – and combining it with rapid oligonucleotide synthesis and episome assembly(5,6), and rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections(1,7,8) – we anticipate it will be possible to synthesize entire E. coli genomes, from functional designs, in less than 2 months.