Over the last few decades, biology research has expanded from the study of
naturally occurring systems to the engineering of new, synthetic functions of our
choosing. This has been possible because of our detailed knowledge of biological
systems, high resolution experimental biology tools, powerful computational resources
and increasingly affordable de novo DNA synthesis technologies. For example, entire
microbial genomes have chemically synthesized and interesting synthetic systems have
been built with novel gene expression dynamics. Additionally, cells have been
engineered to efficiently produce high value compounds, including a number of
recombinant protein molecules. Now, wonderful opportunities abound for improving the
human condition with synthetic biology.
The work presented in this thesis introduces two new synthetic biological
systems. One system focuses on gene expression control while the second is a new
approach to producing and delivering antimicrobial molecules.
The first project is a set of synthetic transcription activators called prokaryotic-
TetOn and prokaryotic-TetOff, that upregulate gene expression in response to
anhydrotetracycline. The molecular geometries of the prokaryotic-TetOn and
prokaryotic-TetOff systems were first optimized using protein structure refinement and
homology modeling. Next, the molecular devices were built and tested experimentally.Finally, both systems were characterized using stochastic models of gene expression.
Prokaryotic-TetOn and prokaryotic-TetOff are the first prokaryotic devices of their kind,
inducible synthetic activators of protein expression. Notably, they have also been designed to serve as firm stepping stones for developing a library of related synthetic
transcription factors and networks.
In a second, distinct project, we considered tacking the extensive use of
antibiotics in agriculture. This overuse appears to have contributed to the emergence of
antibiotic resistant pathogens that are critical to human health. This public health
challenge motivated the development of antibiotic alternatives for agricultural
applications. We engineered lactic acid bacteria to inducibly express and secrete
antimicrobial peptides with activity against Escherichia coli and Salmonella typhimurium
and infantis. This is the first demonstration of bacteria engineered to inducibly produce
peptides with strong activity against Gram-negative pathogens. These systems may also
be used as a foundation for developing a next generation of recombinant bacteria that
produce and deliver antimicrobial peptides.
University of Minnesota Ph.D. dissertation. May 2013. Major: Chemical Engineering. Advisor: Yiannis Kaznessis. 1 computer file (PDF); ix, 111 pages.
Völzing, Katherine Giovanna.
Gene expression control and antimicrobial peptide production by haute couture bacteria.
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