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dc.contributor.advisorPosewitz, Matthew C.
dc.contributor.authorVogler, Brian W.
dc.date.accessioned2018-10-16T16:27:31Z
dc.date.accessioned2022-02-03T13:15:21Z
dc.date.available2019-04-16T16:27:31Z
dc.date.available2022-02-03T13:15:21Z
dc.date.issued2018
dc.identifierVogler_mines_0052E_11585.pdf
dc.identifierT 8596
dc.identifier.urihttps://hdl.handle.net/11124/172561
dc.descriptionIncludes bibliographical references.
dc.description2018 Fall.
dc.description.abstractNannochloropsis is a genus of eukaryotic microalgae that grows well in outdoor bioreactors and produces high yields of triacylglycerols (TAGs), which can be processed into biodiesel. In this work, we chemically characterize the storage carbohydrate of Nannochloropsis for the first time, and interrupt enzymes required for its biosynthesis to (1) understand their function and (2) interrogate whether this unused biomass component can be eliminated without significant impact on viability. To generate targeted gene knockouts, we developed CRISPR/Cas9 methods for Nannochloropsis and first interrupted nitrate reductase, a common target for genetic knockout because it is both inessential and easily scored. The method and validated chassis strain was then used to interrogate the beta-glucan synthase (BGS) and transglycosylase (TGS) enzymes believed to be responsible for the backbone polymerization and branching of the storage beta glucan, respectively. We identified no significant growth defects in our laboratory culturing conditions but did confirm that both genes were fundamental to synthesis of this beta glucan storage carbohydrate. The generated knockouts of either gene do not produce the elevated carbohydrate phenotype of wild-type cells in response to nitrogen deprivation. We also observed a non-bleaching phenotype in knockouts of BGS, where chlorophyll and carotenoid content remain elevated in mature cultures when wild-type cultures reduce their chlorophyll and carotenoid content. The design and diagnostic CRISPR/Cas9 methods developed for Nannochloropsis were then modified to transform a fast-growing high-light-, high-heat-, and high-salt-tolerant microalga Chlorella sp. strain CCMP252 with complexed Cas9-sgRNA to generate insertional knockouts of the nitrate reductase gene, demonstrating the broad applicability of the lessons learned.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado School of Mines. Arthur Lakes Library
dc.relation.ispartof2018 - Mines Theses & Dissertations
dc.rightsCopyright of the original work is retained by the author.
dc.subjectbioengineering
dc.subjectCas9
dc.subjectalgae
dc.subjectCRISPR
dc.subjectcarbohydrate
dc.titleDevelopment of CRISPR/Cas9 in Nannochloropsis and other algae toward understanding and manipulating energy allocation
dc.typeText
dc.contributor.committeememberBoyle, Nanette R.
dc.contributor.committeememberRichards, Ryan
dc.contributor.committeememberTrewyn, Brian
dcterms.embargo.terms2019-04-16
dcterms.embargo.expires2019-04-16
thesis.degree.nameDoctor of Philosophy (Ph.D.)
thesis.degree.levelDoctoral
thesis.degree.disciplineChemistry
thesis.degree.grantorColorado School of Mines
dc.rights.accessEmbargo Expires: 04/16/2019


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