Degenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichment

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dc.contributor.authorFreedman, Benjamin Gordonen
dc.contributor.committeechairSenger, Ryan S.en
dc.contributor.committeememberBarone, Justin R.en
dc.contributor.committeememberPilot, Guillaumeen
dc.contributor.committeememberZhang, Chenmingen
dc.contributor.departmentBiological Systems Engineeringen
dc.date.accessioned2016-06-15T06:00:12Zen
dc.date.available2016-06-15T06:00:12Zen
dc.date.issued2014-12-22en
dc.description.abstractCombinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit.en
dc.description.degreePh. D.en
dc.format.mediumETDen
dc.identifier.othervt_gsexam:4035en
dc.identifier.urihttp://hdl.handle.net/10919/71346en
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectMetabolic Engineeringen
dc.subjectGenomic DNA Libraryen
dc.subjectDegenerate Oligonucleotide Primed PCRen
dc.subjectWhole Genome Amplificationen
dc.subjectRaman Spectroscopyen
dc.subjectClostrdium cellulolyticumen
dc.titleDegenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichmenten
dc.typeDissertationen
thesis.degree.disciplineBiological Systems Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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