RESUMO
The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and is not copied by host polymerases but is selectively copied by an orthogonal DNA polymerase (O-DNAP), which does not copy the genome. We designed mutant O-DNAPs that selectively increase the mutation rate of the orthogonal replicon by two to four orders of magnitude. We demonstrate the utility of our system for accelerated continuous evolution by evolving a 150-fold increase in resistance to tigecycline in 12 days. And, starting from a GFP variant, we evolved a 1000-fold increase in cellular fluorescence in 5 days.
Assuntos
Replicação do DNA , Evolução Molecular Direcionada , Proteínas de Escherichia coli , Escherichia coli , Evolução Molecular , Replicon , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Evolução Molecular Direcionada/métodos , Proteínas de Fluorescência Verde/genética , Tigeciclina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , FluorescênciaRESUMO
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 (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 crossovers1,4 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 synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.
Assuntos
Cromossomos Artificiais Bacterianos , DNA , Escherichia coli , Genoma Bacteriano , Biologia Sintética , Humanos , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Biologia Sintética/métodos , Cromossomos Artificiais Bacterianos/genética , Éxons , Íntrons , Quadruplex G , Elementos Nucleotídeos Longos e Dispersos/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Oligodesoxirribonucleotídeos/biossíntese , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Fatores de TempoRESUMO
The near-universal genetic code defines the correspondence between codons in genes and amino acids in proteins. We refactored the structure of the genetic code in Escherichia coli and created orthogonal genetic codes that restrict the escape of synthetic genetic information into natural life. We developed orthogonal and mutually orthogonal horizontal gene transfer systems, which permit the transfer of genetic information between organisms that use the same genetic code but restrict the transfer of genetic information between organisms that use different genetic codes. Moreover, we showed that locking refactored codes into synthetic organisms completely blocks invasion by mobile genetic elements, including viruses, which carry their own translation factors and successfully invade organisms with canonical and compressed genetic codes.
