A Bacterial Toolkit for Rapid Prototyping of Multicistronic Genetic Circuits from Interchangeable Parts

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dc.contributor.advisorBennett, Matthew R.en_US
dc.creatorBhakta, Shyam Pravinen_US
dc.date.accessioned2024-08-30T15:54:08Zen_US
dc.date.created2024-08en_US
dc.date.issued2024-05-17en_US
dc.date.submittedAugust 2024en_US
dc.date.updated2024-08-30T15:54:08Zen_US
dc.descriptionEMBARGO NOTE: This item is embargoed until 2026-08-01en_US
dc.description.abstractBacteria are the most popular chassis for synthetic biology and industrial biotechnology, owing to the comparative ease of their genetic engineering toward desired functions. Synthetic biology has enabled this goal through systematic gene expression control, by abstracting gene regulatory elements as plug and play modules, including promoters, ribosome binding sites, protein-coding sequences, and terminators. These parts are not perfectly modular and frequently fail to function as predicted in a gene or circuit, owing to compositional effects, chassis environment, etc. Furthermore, the specific parts needed to meet a circuit’s performance criteria are often unknown. Rapid prototyping of DNA is necessary to eliminate bottlenecks in the design-build-test cycle of fast-growing bacteria, and physical standardization and sharing of genetic parts and modules across research labs is a promising solution. The Bacterial Toolkit hierarchical DNA assembly system developed in this thesis and the companion part collection allows complete specification of transcriptional and translational cassette elements in custom combinations of mono- and polycistronic operons in any orientation, to efficiently build bacterial genetic circuits, pathways, and vectors for diverse needs from physically interchangeable parts. A distribution of modern gene expression parts is also provided, including promoters, insulators, insulated ribosome binding sites, transcription factors, protein tags, strong terminators, common E. coli and broad-host range origins, selection markers, plasmid maintenance factors, and E. coli genomic integration homologies. The Bacterial Toolkit is already in use by researchers and has been used for the construction of numerous genetic circuits with functions spanning bacterial pattern formation and population control, asymmetric plasmid partitioning, RNA logic, gene expression library construction, glucose biosensing, bioremediation, protein biomaterials, and environmental monitoring of horizontal gene transfer. Here, we used the Toolkit to demonstrate high assembly fidelity and streamlined genomic integration, test transcriptional insulation afforded by connector elements, investigate the design strategies for UTR insulation in operons, and measure the effects of gene compositional contexts on promoter performance.en_US
dc.embargo.lift2026-08-01en_US
dc.embargo.terms2026-08-01en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationBhakta, Shyam Pravin. A Bacterial Toolkit for Rapid Prototyping of Multicistronic Genetic Circuits from Interchangeable Parts. (2024). PhD diss., Rice University. https://hdl.handle.net/1911/117767en_US
dc.identifier.urihttps://hdl.handle.net/1911/117767en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectSynthetic biologyen_US
dc.subjectDNA assemblyen_US
dc.subjectsynthetic plasmidsen_US
dc.subjectgenetic circuitsen_US
dc.subjectbroad-host vectorsen_US
dc.subjectpromoter contexten_US
dc.subjectgene syntaxen_US
dc.subjecttranscriptional insulationen_US
dc.subjectGolden Gate assemblyen_US
dc.subjectbacterial engineeringen_US
dc.titleA Bacterial Toolkit for Rapid Prototyping of Multicistronic Genetic Circuits from Interchangeable Partsen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentSystems, Synthetic and Physical Biologyen_US
thesis.degree.disciplineSynthetic Biologyen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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