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dc.contributor.advisorRichter, Mark L.
dc.contributor.authorBishop, Stephanie Cara
dc.date.accessioned2009-08-31T02:16:47Z
dc.date.available2009-08-31T02:16:47Z
dc.date.issued2009-05-21
dc.date.submitted2009
dc.identifier.otherhttp://dissertations.umi.com/ku:10232
dc.identifier.urihttp://hdl.handle.net/1808/5448
dc.description.abstractAn inverse polymerase chain reaction (PCR) was employed to construct an engineered F1-ATPase by means of inserting the repressor of primer (Rop) DNA sequence into the region of the ATP synthase gamma (γ) subunit DNA sequence encoding a regulatory dithiol-containing domain. A two-step PCR approach was developed to insert two unusually long (>100 base pairs each) primers encoding 189 base pairs of exogenous DNA into a single site within a pACYC multiple cloning host vector. The construct was verified by means of DNA sequencing. This approach allowed direct insertion of large pieces of DNA into a host DNA molecule without introducing restriction enzyme sites, thus avoiding common shortcomings such as inclusion or omission of base pairs that were associated with traditional subcloning methods. The engineered gamma subunit was designed for assembly with the recombinant alpha (α) and beta (β) subunits into a core F1-ATPase. The rigid twisted helical structure of the Rop protein extended the regulatory domain of the gamma subunit by approximately 60 Ångstroms, thus creating a rigid, rotating armature within the enzyme. The armature is intended for use as a site for attachment of gold particles to monitor rotation of the gamma subunit during ATP hydrolysis.
dc.format.extent44 pages
dc.language.isoEN
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectMolecular biology
dc.subjectAtp synthase
dc.subjectCloning
dc.subjectGamma subunit
dc.subjectInsertion
dc.subjectInverse pcr
dc.subjectRotational kinetics
dc.titleLong Primer Extension by a Novel Inverse PCR Method
dc.typeThesis
dc.contributor.cmtememberDavido, David O.
dc.contributor.cmtememberBenedict, Stephen H.
dc.thesis.degreeDisciplineBiochemistry & Molecular Biology
dc.thesis.degreeLevelM.A.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
kusw.bibid6857577
dc.rights.accessrightsopenAccess


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