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Campus-Only Access for One (1) Year

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Chemical Engineering

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Master of Science in Chemical Engineering (M.S.Ch.E.)

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Improving the ability for probiotic bacteria to adhere to the intestinal wall and form biofilms is critical to promoting a healthful gut environment in patients suffering from inflammatory bowel diseases (IBDs). One probiotic bacterium, Escherichia coli Nissle 1917 (EcN), has been identified as a safe treatment for patients with IBDs but is not able to colonize all treated individuals even with repeated daily doses. In order to enhance its persistence and probiotic capacity, a greater understanding of the relationship between genetics and the potential for bacteria to adhere to surfaces and form biofilms is required.

Toward this end, here a CRISPR-based screen was chosen to explore the underlying functional genomics of EcN because of its exceptional ability to select gene targets with high specificity reducing the number of off-target effects. A strain of EcN was designed to contain a CRISPR interference (CRISPRi) genetic system that can be easily programmed to selectively repress genes. This platform strain would allow for later assays that could uncover the relationship of repression of certain genes with biofilm formation. The CRISPRi system consists of a catalytically dead Cas protein, Lb-dCas12a that is genomically integrated along with a plasmid expressing the guide RNA and was designed here to enable genome-wide CRISPRi screening. The insertions of eYFP into the recA, lacZ, and mutS sites of EcN was carried out using λ-Red recombineering, and its inducible expression was assayed by flow cytometry. For genome-wide CRISPRi screening in EcN, the current design is predicted to be able to target 95.6% of EcN’s annotated genes. Hopefully, this strain will be used in a future CRISPRi screen to bring a more resolved understanding to the genetic controls for biofilm formation in Escherichia coli Nissle 1917.

First Advisor

Lauren Andrews

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Saturday, February 01, 2025