The evolution and engineering of T7 RNA polymerase

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dc.contributor.advisorEllington, Andrew D.en
dc.contributor.committeeMemberAlper, Halen
dc.contributor.committeeMemberBarrick, Jeffreyen
dc.contributor.committeeMemberBull, Jamesen
dc.contributor.committeeMemberFast, Walteren
dc.creatorMeyer, Adam Joshuaen
dc.date.accessioned2015-09-10T14:25:43Zen
dc.date.accessioned2018-01-22T22:28:06Z
dc.date.available2018-01-22T22:28:06Z
dc.date.issued2014-08en
dc.date.submittedAugust 2014en
dc.date.updated2015-09-10T14:25:43Zen
dc.descriptiontexten
dc.description.abstractT7 RNA polymerase is a single protein capable of driving transcription from a simple promoter in virtually any context. This has made it a powerful tool in a range of biotechnology applications. In this work, previous efforts to evolve or engineer T7 RNA polymerase are reviewed. This work is then expanded upon, first with the development of a method for the cell-free evolution of T7 RNA polymerase based on the functioning of an autogene. The autogene is a transcriptional feedback circuit in which active T7 RNA polymerase proteins transcribe their own gene, resulting in exponential amplification of their genetic information. While this system is doomed by an error catastrophe, this can be delayed by the use of in vitro compartmentalization. In response to the limits of the autogene, a novel directed evolution approach termed compartmentalized partnered replication (CPR) is presented. CPR couples the in vivo functionality of a gene to its subsequent in vitro amplification by emulsion PCR. The use of CPR to generate a panel of six versions of T7 RNA polymerase, each specific to one of six promoters, is described. Separately, a rational engineering approach, taken to facilitate the high-yield transcription of fully 2′-modified RNA, is detailed. Two sets of mutations to T7 RNA polymerase, previously known to confer thermal stability and enhance promoter clearance respectively, can be used to enhance the activity of existing T7 RNA polymerase mutants that utilize non-standard nucleotides as their substrates. Next, CPR and random mutagenesis is used to populate the functional fitness landscape of T7 RNA polymerase. This neutral drift library is then challenged to increase the processivity of T7 RNA polymerase, enabling long-range transcription. Finally, the lessons that can be learned about T7 RNA polymerase specifically and molecular evolution and protein engineering generally are discussed.en
dc.description.departmentCellular and Molecular Biologyen
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttp://hdl.handle.net/2152/31292en
dc.language.isoenen
dc.subjectMolecular biologyen
dc.subjectProtein engineeringen
dc.titleThe evolution and engineering of T7 RNA polymeraseen
dc.typeThesisen

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