{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/129709"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/129709","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Combatting pathogens through directed evolution, single-cell sequencing, and synthetic immunology","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-05-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-05-01","abstract_has_math":false,"creators":["Bram, Stanley"],"institution":"University of Illinois Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Mehta, Angad P","van der Donk, Wilfred A","Procko, Erik","Jain, Prashant K"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-04-29","date_published":"2025-04-29","updated_at":"2026-07-22T22:25:05Z","subjects":["Synthetic Biology","Antibody Engineering","Directed Evolution","Immunoglobulin","Salmonella","cofactor analogs","single-cell sequencing."],"languages":["en","eng"],"rights":["Copyright 2025 Stanley Bram"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/129709","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Mehta, Angad P","van der Donk, Wilfred A","Procko, Erik","Jain, Prashant K"]},{"key":"dc:creator","label":"Author","values":["Bram, Stanley"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2025-04-29","2025-05"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Synthetic Biology","Antibody Engineering","Directed Evolution","Immunoglobulin","Salmonella","cofactor analogs","single-cell sequencing."]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2025 Stanley Bram"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/129709"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-05-01","The student, Stanley Bram, accepted the attached license on 2025-04-22 at 11:23.","The student, Stanley Bram, submitted this Dissertation for approval on 2025-04-22 at 11:35.","This Dissertation was approved for publication on 2025-04-29 at 18:44.","DSpace SAF Submission Ingestion Package generated from Vireo submission #21887 on 2025-10-19 at 19:53:37","This thesis explores three innovative applications of directed evolution methodologies for pathogen control, each addressing distinct challenges in therapeutic development. Foremost, I created a novel B cell-based evolution platform for therapeutic antibody development by engineering a synthetic regulatory system that mimics natural somatic hypermutation mechanisms. Through clustered regularly interspaced short palindromic repeat (CRISPR) mediated integration of composite regulatory elements, including the IgHV4-34 promoter and intronic enhancer, I established a cell-type specific system capable of directing controlled genetic variation. This platform demonstrates potential not only for antibody engineering but also for broader applications in protein evolution. Second, in collaboration with the United States Department of Agriculture (USDA), I developed enhanced methods for analyzing the porcine immune response to influenza infection through single-cell sequencing of B cell repertoires. This work established comprehensive protocols for isolating and characterizing complete antibody sequences from individual B cells, enabling high resolution analysis of the immunoglobulin repertoire. While this approach provides unprecedented insight into viral evolution and host immunity, throughput and cost considerations currently constrain its application to population-scale surveillance. However, the foundations provided in this work serve as a starting point to streamline functional, personalized RNA-sequencing methods. Third, I engineered the UDP-galactose 4-epimerase (GalE) system in E. coli to utilize synthetic nicotinamide adenine dinucleotide (NAD+) analogs, establishing a platform for vaccine development through controlled bacterial attenuation. Through directed evolution, I successfully generated enzyme variants capable of utilizing the synthetic cofactor nicotinamide isoquinolinone dinucleotide (NQD+) demonstrating altered cofactor specificity in vitro. While biochemical characterization revealed promising catalytic properties, challenges in cellular uptake, cofactor stability, and enzyme half-life currently limit in vivo implementation. Yet still, the establishment of synthetic, orthogonal cofactor redox systems in this work provides a starting point for further optimization, which, when sufficiently resolved, may result in ‘switchable’ chemistries of life that have not yet been explored throughout the evolution of organisms. Together, these approaches demonstrate the versatility of directed evolution in addressing contemporary challenges in vaccine and therapeutic development, while highlighting the importance of considering both molecular and cellular contexts in engineering biological systems. Future work will focus on overcoming identified technical barriers and expanding these platforms for broader therapeutic applications."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Combatting pathogens through directed evolution, single-cell sequencing, and synthetic immunology"]}]}],"canonical_facts":{"dc:contributor":["Mehta, Angad P","van der Donk, Wilfred A","Procko, Erik","Jain, Prashant K"],"dc:creator":["Bram, Stanley"],"dc:date":["2025-04-29","2025-05"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2027-05-01","The student, Stanley Bram, accepted the attached license on 2025-04-22 at 11:23.","The student, Stanley Bram, submitted this Dissertation for approval on 2025-04-22 at 11:35.","This Dissertation was approved for publication on 2025-04-29 at 18:44.","DSpace SAF Submission Ingestion Package generated from Vireo submission #21887 on 2025-10-19 at 19:53:37","This thesis explores three innovative applications of directed evolution methodologies for pathogen control, each addressing distinct challenges in therapeutic development. Foremost, I created a novel B cell-based evolution platform for therapeutic antibody development by engineering a synthetic regulatory system that mimics natural somatic hypermutation mechanisms. Through clustered regularly interspaced short palindromic repeat (CRISPR) mediated integration of composite regulatory elements, including the IgHV4-34 promoter and intronic enhancer, I established a cell-type specific system capable of directing controlled genetic variation. This platform demonstrates potential not only for antibody engineering but also for broader applications in protein evolution. Second, in collaboration with the United States Department of Agriculture (USDA), I developed enhanced methods for analyzing the porcine immune response to influenza infection through single-cell sequencing of B cell repertoires. This work established comprehensive protocols for isolating and characterizing complete antibody sequences from individual B cells, enabling high resolution analysis of the immunoglobulin repertoire. While this approach provides unprecedented insight into viral evolution and host immunity, throughput and cost considerations currently constrain its application to population-scale surveillance. However, the foundations provided in this work serve as a starting point to streamline functional, personalized RNA-sequencing methods. Third, I engineered the UDP-galactose 4-epimerase (GalE) system in E. coli to utilize synthetic nicotinamide adenine dinucleotide (NAD+) analogs, establishing a platform for vaccine development through controlled bacterial attenuation. Through directed evolution, I successfully generated enzyme variants capable of utilizing the synthetic cofactor nicotinamide isoquinolinone dinucleotide (NQD+) demonstrating altered cofactor specificity in vitro. While biochemical characterization revealed promising catalytic properties, challenges in cellular uptake, cofactor stability, and enzyme half-life currently limit in vivo implementation. Yet still, the establishment of synthetic, orthogonal cofactor redox systems in this work provides a starting point for further optimization, which, when sufficiently resolved, may result in ‘switchable’ chemistries of life that have not yet been explored throughout the evolution of organisms. Together, these approaches demonstrate the versatility of directed evolution in addressing contemporary challenges in vaccine and therapeutic development, while highlighting the importance of considering both molecular and cellular contexts in engineering biological systems. Future work will focus on overcoming identified technical barriers and expanding these platforms for broader therapeutic applications."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/129709"],"dc:language":["en","eng"],"dc:rights":["Copyright 2025 Stanley Bram"],"dc:subject":["Synthetic Biology","Antibody Engineering","Directed Evolution","Immunoglobulin","Salmonella","cofactor analogs","single-cell sequencing."],"dc:title":["Combatting pathogens through directed evolution, single-cell sequencing, and synthetic immunology"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:05Z"}