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Narendra Maheshri. Niesner, Bradley (Bradley Joseph) Massachusetts Institute of Technology. Department of Chemical Engineering. Massachusetts Institute of Technology. Department of Chemical Engineering 2013-10-24T17:43:44Z 2013-10-24T17:43:44Z 2013 2013 http://hdl.handle.net/1721.1/81684 860794260 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. 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. by Bradley Niesner. Ph.D. 84 p. eng Massachusetts Institute of Technology 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. http://dspace.mit.edu/handle/1721.1/7582 Chemical Engineering. Using the Cre-loxP system to randomize target gene expression states and generate diverse phenotypes Generation of phenotypic diversity in yeast using promoter inversion through Cre-lox recombination Thesis application/pdf false Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="41e866d6-99f1-44b6-bf80-41865fe5fa42"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>Using the Cre-loxP system to randomize target gene expression states and generate diverse phenotypes</Title> <Subtitle>Generation of phenotypic diversity in yeast using promoter inversion through Cre-lox recombination</Subtitle> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2013</PublicationDate> <Authors> <Author> <DisplayName>Niesner, Bradley (Bradley Joseph)</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://dspace.mit.edu/handle/1721.1/7582</License> <Keyword>Chemical Engineering.</Keyword> <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> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>