Defense Date

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Integrative Life Sciences

First Advisor

Dr. Stephen S. Fong

Second Advisor

Dr. B. Frank Gupton

Third Advisor

Dr. Matthew C. Hartman

Fourth Advisor

Dr. Michael H. Peters

Fifth Advisor

Dr. Allison Johnson

Abstract

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are leveraged to rapidly optimize several parameters of the ex vivo DNA assembly methodology testing lysates from different engineered strains of E. coli, with various buffer components and using titrations of purified cloning enzymes. Finally, in order to complete the cloning suite, a vector expressing the Pyrococcus furiosis (Pfu) DNA polymerase was designed, constructed and expressed in E. coli to create a ‘functionalized lysate’ capable of ex vivo PCR. Not only do we demonstrate ex vivo cloning methodology as a complete cloning package capable of replacing the expensive cloning reagents currently required by synthetic biologists, but also establish ex vivo as an overarching approach for conducting molecular biology.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-7-2015