Designing And Improving A System And Tools For Targeted Mutation In Growing Bacteria

Other Titles
Abstract
Mutagenesis is commonly employed for strain engineering using contrasting site-specific and untargeted approaches. Site-specific approaches require a high level of knowledge at both the DNA and protein levels to implement highly specified genetic changes, while untargeted approaches require more extensive characterization after a desired mutant phenotype is screened or selected from a pool of randomly generated mutants. Localized mutagenesis techniques, such as error-prone PCR, have been developed to confine mutation to a specific region of DNA and offer an attractive compromise between untargeted and site-specific methods. However, inefficiencies in cloning, transformation, and library construction can make such approaches time consuming. We have developed a system for tunable mutagenesis localized to plasmid vectors based on the controlled overexpression of error-prone DNA polymerase IV (Pol IV) in growing Escherichia coli cells. We showed that mutation is elevated and confined to a target vector in our system due to co-expression of a DNA nickase and placement of its recognition site on target vectors. We provide evidence that this system's mechanism involves error-prone replication by Pol IV during the homologous recombination repair process, a mechanism previously proposed for stationary phase Pol IV-dependent mutation. In addition to providing mechanistic understanding, experiments with our system in diverse strain backgrounds helped to identify polB and mutS deletion backgrounds in which mutation was elevated, and confirmed the requirement for a toxin-antitoxin system encoded by the yafNOP genes, located downstream of chromosomal dinB, encoding Pol IV. We also examined the effects of different numbers of nickase recognition sites and the addition of chi sites to our target vectors, leading to increased mutation rates. Finally, we employed another localized mutagenesis technique to optimize our Pol IV and nickase overexpression system, showing that optimal mutation rates are achieved at lower copy numbers when the expression system is plasmid-borne. In the course of designing and improving our system, we developed new techniques to support mutation research. These include a method for placing confidence intervals on ratios of mutation rate estimates using a bootstrapping approach and a high-throughput microplate fluctuation test inspired by fluorescence Miller assays and the Ames II microplate mutagenicity test.
Journal / Series
Volume & Issue
Description
Sponsorship
Date Issued
2016-02-01
Publisher
Keywords
Mutagenesis; Fluctuation Test; Targeting
Location
Effective Date
Expiration Date
Sector
Employer
Union
Union Local
NAICS
Number of Workers
Committee Chair
March,John C
Committee Co-Chair
Committee Member
Peters,Joseph E.
Worobo,Randy W.
Degree Discipline
Agricultural and Biological Engineering
Degree Name
Ph. D., Agricultural and Biological Engineering
Degree Level
Doctor of Philosophy
Related Version
Related DOI
Related To
Related Part
Based on Related Item
Has Other Format(s)
Part of Related Item
Related To
Related Publication(s)
Link(s) to Related Publication(s)
References
Link(s) to Reference(s)
Previously Published As
Government Document
ISBN
ISMN
ISSN
Other Identifiers
Rights
Rights URI
Types
dissertation or thesis
Accessibility Feature
Accessibility Hazard
Accessibility Summary
Link(s) to Catalog Record