Synthetic biology increasingly depends on the ability to construct, maintain, and verify DNA molecules that encode complex biological functions. Many of the most valuable genetic programs, however, are difficult to propagate in conventional cloning hosts because the same cells used to amplify the DNA are also exposed to the functions encoded by that DNA. This coupling is especially problematic for toxic genes, antibacterial proteins, lysis genes, nucleases, and complete bacteriophage genomes, where the desired activity can damage or kill the host and select for mutants that have lost the intended function. This thesis develops a synthetic-chassis strategy for separating DNA amplification from functional gene expression. Using a bacterial host with a refactored genetic code, natural-rule coding sequences containing selected codons can be maintained as DNA while expression of their protein products are silenced. Replicon backbones and selection markers are encoded in mutually compatible rule so that they remain active in the synthetic chassis, allowing toxic cargoes to be propagated, sequence-verified, and transferred into an execution context where the standard decoding rule restores function. This framework is applied to the construction of otherwise difficult DNA, including toxic genes and bacteriophage genomes, and is extended toward the assembly of increasingly complex phage systems. The thesis also presents SynPl-Seq, a rapid colony-to-consensus workflow for whole-plasmid sequence verification. By combining backbone-specific whole-plasmid PCR, multiplexed barcoding and Nanopore sequencing, SynPl-Seq enables high-throughput validation of candidate clones within a single working day and supports the iterative construction workflows required for large or unstable genetic systems. Together, these studies advance a central concept: the genetic code can be used not only to expand or contain biological function, but also to route when and where a genetic program is expressed. Refactored-code chassis provide a protected environment for constructing DNA whose activity must be preserved but temporarily silenced, offering a general platform for phage engineering, toxic-cargo cloning, and future biological systems that require context-dependent expression control.
Jianyi Huang (Wed,) studied this question.