Design and Application of Regulatory-Protein-Based Biosensors

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dc.contributor.committeeMemberCirino, Patrick C.
dc.contributor.committeeMemberVaradarajan, Navin
dc.contributor.committeeMemberConrad, Jacinta C.
dc.contributor.committeeMemberYeo, Hye-Jeong
dc.contributor.committeeMemberFox, George E.
dc.creatorWang, Zhiqing
dc.date.accessioned2018-11-30T18:26:19Z
dc.date.available2018-11-30T18:26:19Z
dc.date.createdMay 2018
dc.date.issued2018-05
dc.date.submittedMay 2018
dc.date.updated2018-11-30T18:26:19Z
dc.description.abstractMetabolic engineering for microbial overproduction of biochemicals benefits from continued advances in directed evolution techniques. Rational design of both proteins and metabolic pathways is often hampered by a lack of sufficient insights into sequence-function relationships. Combinatorial design incorporating random and semi-rational mutagenesis remains a powerful method to explore sequence space when information is limited. While large libraries are often desired in such directed evolution approaches, technical limitations in the screening process often constrain the number of mutants that can be observed. Genetically encoded biosensors can address this problem by correlating the quantity of the biochemical of interest (input signal) to a readily detectable phenotype (including growth), facilitating high-throughput screening of large enzyme or pathway libraries. This thesis describes efforts to streamline the process by which regulatory-protein-based biosensors are designed for desired performances. A (dual) selection/counter-selection system based on expression of the tetracycline resistance protein encoded by tetA was developed and implemented to isolate variants of an AraC-based biosensor for enhanced sensitivity and desired specificity toward triacetic acid lactone (TAL). Equipped with TetA dual-selection, an array of TAL sensors were identified with lowered background signal and improved sensitivity. All these TAL sensors have background expression levels that are lower than 60% of the parent (AraC-TAL1), with the lowest being 1.3 times the background expression level of wild-type AraC. One of the TAL sensors has a sensitivity that is 10 times higher than the original sensor. AraC-TAL1 shows similar responses to both TAL and orsellinic acid (a tetraketide). A TAL sensor that does not respond to orsellinic acid would be useful to engineer PKS substrate elongation channel and/or cyclization specificity. To engineer such a sensor, TetA dual-selection was carried out with orsellinic acid as a decoy. A TAL sensor was isolated with a response to TAL similar to AraC-TAL1, but its response for orsellinic acid is completely abolished. TetA dual-selection provides a robust method for rapid evolution of regulatory-protein-based biosensors. The AraC-based TAL sensors isolated in this research are valuable in engineering PKS activities.
dc.description.departmentChemical and Biomolecular Engineering, Department of
dc.format.digitalOriginborn digital
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/10657/3512
dc.language.isoeng
dc.rightsThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).
dc.subjectBiosensors
dc.subjectRegulatory protein
dc.subjectTriacetic acid lactone
dc.subjectPolyketide
dc.subjectHigh-throughput screening
dc.titleDesign and Application of Regulatory-Protein-Based Biosensors
dc.type.dcmiText
dc.type.genreThesis
local.embargo.lift2020-05-01
local.embargo.terms2020-05-01
thesis.degree.collegeCullen College of Engineering
thesis.degree.departmentChemical and Biomolecular Engineering, Department of
thesis.degree.disciplineChemical Engineering
thesis.degree.grantorUniversity of Houston
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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