ORCID

https://orcid.org/0000-0003-4072-8509

Date of Award

2022

Document Type

Thesis

Degree Name

MS in Biology

Department

Biological Sciences

First Advisor

Javier Juarez

Second Advisor

Dianella Howarth

Third Advisor

Matteo Ruggiu

Abstract

The need for an internal control to govern engineered strain survival is becoming apparent when considering the biosafety and ethics within biotechnological research. Using understudied regulatory elements known as RNA thermometers (RNATs) that can modulate protein translation based on temperature changes and a Type II restriction modification system, we posit the creation of a synthetic temperature-activated epigenetic biocontainment system, termed the ‘sentinel switch’. We have mined a wide variety of bacteria from Yersinia pestis to Synechocystis sp. looking for novel RNATs. A complementary synthetic biology approach based on library generation and screening of chimeric RNATs (ChRNATs) was also conducted. RNATs were then inserted within a pUC19 chassis. RNAT-containing plasmids carrying fluorescent reporter genes were transformed into Escherichia coli NEB5α and MG1655. The functionality of the RNATs inserted in the new vectors was measured through kinetic fluorescence assays. Furthermore, epigenetic components were inserted into our modular system. Our future plans contemplate testing of the functionality of the epigenetic system components via survival assays. A mathematical model was posited for optimization of our sentinel switch and to characterize the dynamics of our collection of natural and synthetic RNATs. The creation of novel kill-switches represent a significant contribution providing an internal level of safety for work with potentially biohazardous agents while spearheading the push for the need of better self-imposed biocontainment strategies in biotechnological research.

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