Master of Science
Edgell, David R.
Karas, Bogumil J.
Biocontainment is an integral part of biomedical research that aims to protect the environment and human health by containing hazardous or invasive organisms in the laboratory. Containment systems often rely on elaborate genetic circuits; however, cells may escape containment by developing mutations that render the genetic circuits inviable or resistant to killing mechanisms. The aim of this thesis was to create a site-specific nuclease for biocontainment of plasmids in the mammalian gastrointestinal tract. LAGLIDADG homing endonucleases would be good candidate nucleases for a biocontainment system as they are resistant to mutations in their coding sequence and their target sequence in comparison to other nucleases, such as Cas9. Screening mutagenic libraries of the I-OnuI nuclease yielded a single variant that displays thermosensitive properties in vivo that may prove useful for biocontainment purposes. Using thermosensitive LAGLIDADG homing endonucleases for this purpose may prove to be a new, more robust approach to biocontainment.
Summary for Lay Audience
Biocontainment is an essential part of biomedical research that aims to protect the environment and human health by containing potentially hazardous organisms in the laboratory. Efforts to develop containment systems often rely on elaborate and expansive DNA circuits. The methods used previously also raise the concern that the cells may be able to escape containment, either through mutations that break the DNA circuits or by the evolution of resistance to killing mechanisms. Nucleases, which are enzymes that cut DNA, are attractive candidates for use as a containment mechanism. DNA cutting proteins that can target internal DNA sequences are called endonucleases (in comparison to exonucleases which target the ends). LAGLIDADG homing endonucleases are small, yet highly specific DNA cutting proteins that are resistant to mutations in their DNA sequence and the particular sequence they cut (in comparison to other DNA cutting proteins). As a result of their resilience and specificity, homing endonucleases from the LAGLIDADG family were chosen as the candidate enzymes for developing a biocontainment mechanism. Screening libraries of LAGLIDADG DNA cutting enzymes yielded a single type that displays temperature-sensitive properties when used in a bacterial cell. Temperature sensitivity is useful as a biocontainment mechanism as it means the bacterial cell can only survive in a specific temperature range. These properties may prove helpful for biocontainment purposes in that it could be used as a new, more straightforward, more robust approach to biocontainment.
Leichthammer, Christopher D., "Development of a Thermosensitive Endonuclease to Act as a Plasmid Kill-Switch" (2020). Electronic Thesis and Dissertation Repository. 7585.
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