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Understanding Microbial Coexistence Via Targeted Gene Knockouts With An Improved CRISPR/Cas9 Assisted Genome Editing Method

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2025-06-13
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This thesis is part of a larger project that aims to understand the fundamental principles governing the coexistence of strains in microbial consortia. The main objective of this thesis was to engineer a library of Escherichia coli strains, each of which could metabolize a prescribed set of carbon sources. To accomplish this, a scarless genetic knockout system that utilizes the CRISPR/Cas9 and the λ red recombination system was employed to obtain targeted gene knockouts. We encountered several difficulties building the library, which led us to improve the scarless genetic knockout system by introducing a fast screening method for false positive transformants, which is the main result of the thesis. Then, we used this improved scarless genetic knockout system to create scarless knockouts of genes involved in the metabolizing maltose (malG, malF, malE, malK, lamB, malM) and galactose (galT, galK, galM), and non-scarless knockouts of genes involved in metabolizing fructose (fruB, fruK,fruA), mannose (manX, manY, manZ) and ribose (rbsD, rbsA, rbsC, rbsB, rbsK). In parallel to engineering these knockout strains, we utilized strains from the E. coli Keio knockout collection to investigate how catabolite repression might affect the study of coexistence criteria in our engineered consortia, and performed a preliminary experiment to test mathematical theories of microbial coexistence.

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2023-05

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Giometto, Andrea

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Barstow, Buz

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Civil and Environmental Engineering

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M.S., Civil and Environmental Engineering

Degree Level

Master of Science

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Government Document

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Attribution-NonCommercial-NoDerivatives 4.0 International

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dissertation or thesis

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