{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/8388"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/8388","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"A two-color bacterial two-hybrid system for selecting sequence-specific DNA-binding proteins via fluorescence activated cell sorting","abstract":"Proteins that recognize specific DNA sequences play a major role in many biological processes, and the ability to select or design novel DNA binding proteins could have a major impact on many areas of biotechnology and medicine. This thesis begins with background information on zinc finger DNA-binding proteins and on methods to select these proteins, and how they can be used to regulate endogenous human genes. Next, I describe a structural and biochemical study of a DNA-binding protein which demonstrates some of the complexities of the protein-DNA interface, and which highlights difficulties for designing sequence-specific proteins via a simple code. I then develop an experimental system (starting with a pre-existing bacterial two-hybrid selection system) which allows the selection of proteins based on their preference of one DNA site over another. I use this system to attempt to attenuate the affinity of a zinc finger protein without destroying its specificity. Finally, I describe an experiment in which I select a new domain that adds sequence specificity to a pre-existing protein from a library of completely random peptides.","abstract_html":"Proteins that recognize specific DNA sequences play a major role in many biological processes, and the ability to select or design novel DNA binding proteins could have a major impact on many areas of biotechnology and medicine. This thesis begins with background information on zinc finger DNA-binding proteins and on methods to select these proteins, and how they can be used to regulate endogenous human genes. Next, I describe a structural and biochemical study of a DNA-binding protein which demonstrates some of the complexities of the protein-DNA interface, and which highlights difficulties for designing sequence-specific proteins via a simple code. I then develop an experimental system (starting with a pre-existing bacterial two-hybrid selection system) which allows the selection of proteins based on their preference of one DNA site over another. I use this system to attempt to attenuate the affinity of a zinc finger protein without destroying its specificity. Finally, I describe an experiment in which I select a new domain that adds sequence specificity to a pre-existing protein from a library of completely random peptides.","abstract_has_math":false,"creators":["Miller, Jeffrey Christopher, 1974-"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Dept. of Biology.","school":null,"contributors":[],"advisors":["Carl O. Pabo."],"committee_chairs":[],"committee_members":[],"year":2002,"date_issued":"2002","date_published":"2002","updated_at":"2026-07-22T22:20:59Z","subjects":["Biology."],"languages":["eng"],"rights":["M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. 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