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Journal Article
Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly
2023
Overview
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 precursors 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 developed 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
, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections
1
,
7
,
8
, we anticipate that it will be possible to synthesize entire
E. coli
genomes from functional designs in less than 2 months.
BAC stepwise insertion synthesis (BASIS) can be used to build synthetic genomes for diverse organisms, and continuous genome synthesis (CGS) enables the rapid synthesis of entire
Escherichia coli
genomes from functional designs.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ 13/31
/ 42/109
/ 42/35
/ 42/44
/ 45/23
/ 45/56
/ 45/77
/ Assembly
/ Bacterial artificial chromosomes
/ Chromosomes, Artificial, Bacterial - genetics
/ Cloning
/ Design
/ DNA
/ E coli
/ Exons
/ Genome, Bacterial - genetics
/ Genomes
/ Humanities and Social Sciences
/ Humans
/ Introns
/ Long Interspersed Nucleotide Elements - genetics
/ Methods
/ Oligodeoxyribonucleotides - biosynthesis
/ Oligodeoxyribonucleotides - genetics
/ Oligodeoxyribonucleotides - metabolism
/ Repetitive Sequences, Nucleic Acid - genetics
/ Science
/ Short interspersed nucleotide elements
