Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli.

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.

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 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.

Refactored genetic codes enable bidirectional genetic isolation.

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.

High-Content Screening and Computational Prediction Reveal Viral Genes That Suppress the Innate Immune Response.

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single-virus or -gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human virus genes. We found that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3). We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCE Infectious diseases caused by viral pathogens exacerbate health care and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large-scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study, we assembled a large viral-gene library from diverse viral families and developed a high-content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step toward early-stage evaluation of emerging and understudied viruses.