Hierarchical in vivo assembly for rapid and high-throughput construction of long DNA sequences - ABSTRACT Advances in synthetic biology, biological chemistry, genomic engineering, and drug discovery increasingly rely on constructing long and complex DNA sequences. Applications such as engineering biosynthetic pathways, designing synthetic chromosomes, and optimizing combinatorial genetic libraries require efficient and reliable assembly methods for DNA constructs exceeding 20kb. While existing technologies like Gibson and Golden Gate Assembly are effective for smaller constructs, they face challenges with longer and more complex sequences. Yeast-based assembly, though capable of handling larger constructs, is limited by low yields, long turnaround times, and scalability constraints. To address these challenges, BacStitch DNA has developed a novel in vivo DNA assembly platform in E. coli. This platform integrates bacterial conjugation, in vivo DNA cutting, and homologous recombination, enabling high-throughput, seamless, and modular assembly of constructs exceeding 80kb. By eliminating in vitro steps, sequence scars, and the need for refactoring, the platform reduces costs and accelerates the design-build-test cycle. Key features include modular workflows that allow building blocks to be reused and shuffled without PCR and barcoded arrays for high-throughput sequence validation. In this project, we aim to enhance the platform by transitioning from sequential to hierarchical assembly workflows. This extension will enable seamless transitions between intermediate constructs and final assemblies, significantly improving speed, scalability and efficiency. Specific Aim 1 focuses on developing, optimizing, and scaling the hierarchical in vivo DNA assembly platform. By integrating orthogonal conjugation systems with conditional origins of replication, the platform will streamline workflows, reduce hands-on time, and enhance the accuracy and throughput of DNA assembly. Constructs exceeding 100kb, including those with complex sequence features, will be efficiently assembled. Specific Aim 2 will demonstrate the platform’s utility by constructing biosynthetic gene clusters (BGCs) encoding polyketide synthases and nonribosomal peptide synthetases. These clusters, which often include repetitive sequences and high GC content, encode valuable products such as antibiotics, immunosuppressants, and anticancer agents. The platform will facilitate the reuse, shuffling, and optimization of genetic elements, accelerating the development of novel derivatives and improving pathway production in heterologous hosts. This project will deliver an innovative DNA assembly technology that reduces costs, enhances scalability, and expands the range of accessible DNA sequences. By enabling the rapid construction of long and complex constructs, this platform will empower researchers and developers across synthetic biology, genomics, and biopharmaceutical industries to address challenging projects, driving innovation and discovery.
