Browsing by Subject "Genetic devices"

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    Gene expression control and antimicrobial peptide production by haute couture bacteria
    (2013-05) Völzing, Katherine Giovanna
    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.