Megabase-Scale Genome Synthesis: Continuous Assembly of E. coli Sections and Human DNA at Unprecedented Scale

Building synthetic genomes at the scale required for whole-organism engineering demands new DNA assembly methods. This 2023 Nature paper from the Chin group introduced two technologies, BASIS and CGS, that substantially accelerate the construction of megabase-scale synthetic DNA, advancing the infrastructure needed for genome-recoded organisms.

BASIS (BAC stepwise insertion synthesis) is a method for assembling megabase-scale DNA in E. coli episomes, circular DNA elements maintained alongside the chromosome. The team used BASIS to assemble 1.1 megabases of human genomic DNA, containing exons, introns, repetitive sequences, G-quadruplexes, and both long and short interspersed nuclear elements (LINEs and SINEs). This demonstrated that BASIS can handle the full structural complexity of eukaryotic genomes, not just the relatively simpler bacterial sequences.

CGS (continuous genome synthesis) addresses the second bottleneck: replacing the native genome of a living organism with synthetic DNA. CGS continuously replaces sequential 100-kilobase stretches of the E. coli genome with synthetic DNA, minimising recombination between synthetic and native sequences. The output of each 100 kb replacement becomes the input for the next, without requiring sequencing between steps. Using CGS, the team synthesised a 0.5 Mb section of the E. coli genome, a key intermediate in its total synthesis, from five episomal constructs in ten days.

For Constructive Bio, these technologies represent the engineering pipeline that enables the creation and iteration of genome-recoded strains. Building the next generation of production organisms, including strains with additional freed codons, optimised expression characteristics, or expanded ncAA repertoires, depends on the ability to synthesise and install large genomic segments rapidly and reliably. BASIS and CGS are what make this scalable.