Continuous genome synthesis and megabase human DNA assembly in E. coli
Whole genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably, and in parallel we need: i) methods for assembling Mbp of DNA from shorter pre-cursors, and ii) strategies for rapidly and scalably replacing genomic DNA with synthetic DNA. Here we develop BAC Stepwise Insertion Synthesis (BASIS), a method for Mbp-scale assembly of DNA in E. coli episomes. We use BASIS to assemble 1.1 Mbp of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes and LINE/SINE elements. 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 kbp stretches of the E. coli genome with synthetic DNA from episomes; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kbp replacement provides, without sequencing, the input for the next 100 kbp replacement.  Using CGS, we synthesize a 0.5 Mbp section of the E. coli genome – a key intermediate in its total synthesis1 – from five episomes in ten days. By parallelizing CGS – and combining it with rapid oligonucleotide synthesis and episome assembly5,6, and rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8 – we anticipate it will be possible to synthesize entire E. coli genomes, from functional designs, in less than two months.