Multiplexed in vivo assembly of long and complex DNA

PROJECT SUMMARY/ ABSTRACT High-throughput, low-cost synthesis of long and complex DNA could open up new frontiers in synthetic biology, drug development, and genomics by facilitating the production and characterization of previously inaccessible genes and genetic pathways. Microarray printing technologies provide for high-throughput and low-cost synthesis of almost any oligonucleotide up to 300 bases long, while new technologies, such as enzymatic synthesis, have the potential to synthesize sequences de novo up to 2kb. To construct 2-5kb DNA blocks, oligonucleotides can be assembled using a suite of in vitro technologies, including polymerase cycling assembly, Gibson assembly, and Golden Gate assembly. However, application of in vitro technologies to assemble DNA longer than ~2-5kb requires considerable hands-on time, is difficult to multiplex and scale, is unreliable for construction of complex DNA (e.g. extreme GC content, homopolymers, repeats, DNA structure), and is extremely challenging for DNA longer than ~7kb. In addition, current methods to screen assemblies for sequence perfect clones are expensive and require considerable hands-on time or complex robotics. To overcome these hurdles, we have developed a novel, low-cost, highly multiplexed in vivo method to assemble long and complex DNA, and to sequence verify clones. In this method, arrays of DNA blocks are introduced into plasmids and bacteria, and these arrays are stitched together sequentially by bacterial mating. Following assembly, bacterial arrays are barcoded via another round of bacterial mating, enabling massively parallel DNA isolation and sequencing library preps from pooled clones. Compared to existing in vitro cloning our approach for stitching either oligos or DNA blocks together is up to 1 00X cheaper and requires up to 100X less hands-on time than current technologies, with no increase in total time. Importantly, in vivo assembly can assemble, at scale, DNA that is at least 15kb and that contains many regions of complex DNA. Here, we propose to harden, scale, and commercialize this technology by i) building a high-throughput production pipeline for in vivo DNA assembly and sequence validation, and ii) building a sequence analysis pipeline and database that will aid in both tracking and predicting error modes of in vivo DNA assembly. These aims will enable BacStitch to initially provide custom services and eventually standardized products that fulfill an unmet need for long and complex DNA.