Dissertation/Thesis Abstract

Construction and Characterization of Genomically Recoded Organisms and Synthetic Auxotrophs
by Rovner, Alexis Jennifer, Ph.D., Yale University, 2015, 244; 3663588
Abstract (Summary)

There are two important features that characterize the canonical genetic code. First, the genetic code is redundant. There are 64 three-letter words that can be assembled from four letters (43), but only 20 types of amino acids are translated. Combinations of only 20 chemistries have been more than sufficient to generate the amazing diversity of life. However, if we could minimize this redundancy to encode additional nonstandard amino acids (NSAAs), this could permit the evolution of entirely new biological functions and possibly, new organisms.

Second, the genetic code is conserved. All species utilize the same biochemical foundation to sustain life. This conservation permits organisms to share beneficial traits via horizontal gene transfer (4) and enables accurate expression of heterologous genes in non-native organisms (10). However, the conservation of the genetic code also allows viruses to hijack host translation machinery (15) and compromise cell viability. It further allows unwanted sharing of information between genetically modified organisms (GMOs) and their environment. Virus resistance and intrinsic biocontainment – biological barriers limiting the spread and survival of microorganisms in natural environments – are among today's major unsolved problems in biotechnology.

Changing the genetic code to create genomically recoded organisms (GROs, whose codons have been reassigned to create an alternate genetic code) could solve these challenges. The focus of my graduate work was to construct and characterize GROs that: 1) provide dedicated codons to enable sustained incorporation of more than 20 amino acids as part of an expanded genetic code, and 2) depend on synthetic biochemical building blocks for viability, to advance orthogonal barriers between organisms and their environment.

Chapter 2. Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of an orthogonal translation system (OTS) consisting of an engineered elongation factor and archaeal aminoacyl-tRNA synthetase (aaRS)/tRNA pair to the normal E. coli translation machinery (16). However, protein yield suffered horn expression of the OTS and competition with release factor 1 (RF1). In a strain lacking RF1, phosphoserine phosphatase, and where only seven essential TAG codons were converted to TAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.

Chapter 3. To create a GRO we replaced all known TAG stop codons in E. coli with synonymous TAA codons, which permitted the deletion of RF1 and reassignment of UAG translation function. This GRO exhibited improved cellular fitness and improved properties for incorporation of NSAAs that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Chapter 4. GMOs are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals ( 17). Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems (18, 19), which include bioremediation (20) and probiotics (21). Here, we describe the construction of a series of GROs (22) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced in-frame TAG codons into 22 essential genes of a GRO containing an OTS, linking their expression to the incorporation of sAAs. Of the 60 variants isolated, notable strains were not rescued by environmental cross-feeding, maintained robust growth, and exhibited undetectable escape frequencies upon culturing ∼10 11 cells in liquid media for 20 days, a significant improvement over existing biocontainment approaches (18, 19, 23–27).

Indexing (document details)
Advisor: Isaacs, Farren
Commitee:
School: Yale University
School Location: United States -- Connecticut
Source: DAI-B 76/11(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Genetics, Biomedical engineering
Keywords: Biological containment, Genetic code, Genomically recoded organisms, Protein engineering, Synthetic biology, Unnatural amino acids
Publication Number: 3663588
ISBN: 9781321946208
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