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.
\r\n\r\nThis 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.
\r\n\r\nThe 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.
\r\n\r\nTogether, 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.
", "doi": "10.7907/dd90-sp96", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18700", "collection": "thesis", "collection_id": "18700", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292026-200819986", "primary_object_url": { "basename": "PhD-Thesis-RJ-Chadha-Final.pdf", "content": "final", "filesize": 69956436, "license": "other", "mime_type": "application/pdf", "url": "/18700/1/PhD-Thesis-RJ-Chadha-Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Stimulated Raman Imaging for Spatial Metabolomics: From Metabolite Mapping to Cellular Function", "author": [ { "family_name": "Chadha", "given_name": "Rahuljeet Singh", "orcid": "0000-0002-3805-6144", "clpid": "Chadha-Rahuljeet-Singh" } ], "thesis_advisor": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "thesis_committee": [ { "family_name": "Semlow", "given_name": "Daniel R.", "orcid": "0000-0001-6538-9713", "clpid": "Semlow-D-R" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Karthikeyan", "given_name": "Smruthi", "orcid": "0000-0001-6226-4536", "clpid": "Karthikeyan-Smruthi" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metabolism encompasses the full network of chemical reactions sustaining life- the creation, utilization, and storage of biologically active small molecules known as metabolites. Understanding how these molecules are organized in space and real time, at the subcellular level, has long been a fundamental bottleneck in advancing our knowledge of cellular metabolism. In recent years, stimulated Raman scattering (SRS) microscopy has emerged as a powerful approach to visualize metabolism directly in living systems, offering high sensitivity, chemical selectivity, and a non-destructive imaging modality uniquely suited to longitudinal studies. Despite these attributes, metabolic imaging with SRS remains in its early stages relative to the well-established fluorescence-based and mass spectrometry imaging methods that dominate the field.
\r\n\r\nTherefore, this thesis aims to push the boundaries of SRS microscopy toward quantitative, spatially resolved metabolomics to enable direct visualization of metabolic activity in living systems. By applying emerging Raman probes across diverse biological systems, this work reveals metabolic vulnerabilities that deepen insight into disease mechanisms and can inform therapeutic development.
\r\n\r\nIn Chapter 1, we introduce metabolic imaging using SRS microscopy and underscore its unique advantages for longitudinal imaging in living biosystems. We cover both label-free (untargeted) and labeled (targeted) approaches using SRS to probe metabolism in situ. We discuss both the advantages and current limitations of SRS microscopy and propose strategies for integrating it with complementary functional \u201c-omics\u201d techniques to gain deeper insight into cellular metabolism.
\r\n\r\nIn Chapter 2, we explore the use of untargeted SRS microscopy to non-invasively characterize the chemical landscape of engineered living materials (ELMs) in real-time. By correlating spectral data with the mechanical properties of genetically modified biofilms, we find multiscale metabolic heterogeneity within these systems. This approach enables quantitative, real-time monitoring of ELMs that would enable improved design in applications spanning biomedicine, sustainability, and responsive materials.
\r\n\r\nIn Chapter 3, we utilize targeted SRS microscopy to tackle critical questions in cardiovascular health with a focus on understanding the metabolic basis of diabetes. In collaboration with the Chen Lab at City of Hope Medical Center and the TeSlaa lab at UCLA, we observe glycogen accumulation in live endothelial cells under diabetic stress. By imaging glutamine and lactate metabolism using Raman probes for the first time in this system, we reveal how intracellular glycogen pools shape early metabolic adaptations in the endothelium during glucose deprivation- providing new insight into the vascular ramifications of hyperglycemia.
\r\n\r\nIn Chapter 4, we introduce MATRIX-SRS (Metabolic Activity TRacing of the trIcarboXylic acid cycle by Stimulated Raman Scattering microscopy), a platform for spatially resolved, quantitative imaging of TCA cycle activity in live cells. By combining deuterium-labeled metabolic probes with hyperspectral stimulated Raman scattering microscopy, we directly visualize and map TCA-associated metabolism at subcellular resolution. We next integrate density functional theory (DFT) with reaction network modeling to develop a robust in situ quantification pipeline in live cells. Using this approach, we identify a global attenuation of TCA activity during epithelial-to-mesenchymal transition (EMT) and, for the first time, achieve absolute quantification of deuterium-labeled biomass in live cells under native and drug-treated conditions, establishing a general framework for live-cell spatial metabolomics.
\r\n\r\nIn Chapter 5, we present MetaboRamics, highly multiplexed metabolic imaging using stimulated Raman for spatial metabolomics in live cells. Through rational probe design, isotope editing, and robust spectral unmixing, we establish a 16-color metabolic palette spanning key pathways alongside endogenous protein, lipid, and redox signals, with organelle-targeted probes enabling spatial interactomics. We apply this high-content platform to EMT to observe a global metabolic rewiring in mesenchymal cells. We further resolve subcellular adaptations under diverse metabolic stress conditions in live epithelial cells. MetaboRamics enables 16-plex, subcellular metabolomics in living systems and opens new avenues for applications in drug discovery and clinical diagnostics.
\r\n\r\nThrough these studies, I demonstrate the power of stimulated Raman imaging for subcellular, spatiotemporal metabolomics to visualize metabolism in living systems and uncover its roles in both health and disease.
", "doi": "10.7907/n5c3-jw36", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" } ]