{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/81684"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/81684","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Using the Cre-loxP system to randomize target gene expression states and generate diverse phenotypes","abstract":"Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in S. cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between 4 distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites and an additional recombinase should increase this number to at least 30. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of 4 different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre-loxP recombination works in many organisms, this strategy should be readily extendable.","abstract_html":"Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in S. cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between 4 distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites and an additional recombinase should increase this number to at least 30. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of 4 different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre-loxP recombination works in many organisms, this strategy should be readily extendable.","abstract_has_math":false,"creators":["Niesner, Bradley (Bradley Joseph)"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Chemical Engineering.","school":null,"contributors":[],"advisors":["Narendra Maheshri."],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013","date_published":"2013","updated_at":"2026-07-22T22:21:12Z","subjects":["Chemical Engineering."],"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. See provided URL for inquiries about permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/81684","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Narendra Maheshri."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Chemical Engineering."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. 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They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/81684"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.","Title as it appears in MIT Commencement Exercises program, June 2013: Generation of phenotypic diversity in yeast using promoter inversion through Cre-lox recombination. Page 84 blank. Cataloged from PDF version of thesis.","Includes bibliographical references."]},{"key":"dc:description.abstract","label":"Abstract","values":["Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in S. cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between 4 distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites and an additional recombinase should increase this number to at least 30. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of 4 different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre-loxP recombination works in many organisms, this strategy should be readily extendable."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Using the Cre-loxP system to randomize target gene expression states and generate diverse phenotypes"]}]}],"canonical_facts":{"dc:contributor.advisor":["Narendra Maheshri."],"dc:contributor.department":["Massachusetts Institute of Technology. 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