Biochemistry, Department of


Date of this Version

Winter 12-5-2013


Thomas M. Plucinak. Making Chlamydomonas reinhardtii a better model organism: tackling the inefficiency of NUCLEAR transgene expression and improving methods for the generation and characterization of insertional mutant libraries. Dec, 5 2013. University of Nebraska-Lincoln


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Donald P. Weeks. Lincoln, Nebraska: December, 2013

Copyright (c) 2013 Thomas M. Plucinak


The green algal species Chlamydomonas reinhardtii possesses many beneficial features that have made it a useful model organism for many decades. Many types of experimentation however are difficult to conduct with this organism due to the relative under-development of genetic tools available for use. Tasks such as transgene expression, overexpression of proteins of interest (POIs) or site specific genomic modification that are routine in other more facile microbial model organisms such as Escherichia coli and yeast are difficult to accomplish in C. reinhardtii. The second chapter of this thesis describes the development of a novel nuclear transgene expression system that alleviates many of the difficulties associated with transgene expression in C. reinhardtii and in some instances allows for overexpression of POIs. This system relies on viral 2A peptides to link the coding regions of selectable marker genes to those of other transgenes of interest – transgenes that do not confer a selectable phenotype to cells when expressed. Implementation of this system allows for the construction and utilization of polycistronic genes in C. reinhardtii that significantly outperform traditional vectors used for expressing transgenes. Overall, this new tool enhances the ability to genetically engineer the nuclear genome of C. reinhardtii and expands the overall spectrum of experimentation that can be conducted with this organism.

The third chapter of this thesis describes the development of optimized methods for generating C. reinhardtii mutant libraries using insertional mutagenesis and for characterizing the resulting mutants. Because site specific modification of the genome of this organism is extremely inefficient, insertional mutagenesis has necessarily become the most favored methodology for generating mutants that are subsequently used to study various biological systems. Standard insertional mutagenesis procedures however suffer from random insertion of transgenes, resulting in the creation of large populations of mutants bearing insertions in sequences such as intergenic regions that do not contribute significantly to the function of any gene. The optimized insertional mutagenesis method reported here relies on the use of a selectable marker lacking a promoter to generate mutant populations consisting of a large majority of individuals that contain disruptions in transcribed regions. And finally, many of the most common methods that exist for identifying the exact genomic locations of foreign DNA insertion events can be inefficient. To address this deficiency, an optimized version of inverse PCR was developed that allows for the characterization of marker gene insertion events in a highly efficient, facile and cost-effective manner.

Advisor: Donald P. Weeks

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