2022 Theses Doctoral

Expanding Biosensing Capabilities of Engineered Yeast

Crnkovic, Tea

Synthetic biology is an emerging field which has led to development of many useful applications of engineered biological networks and systems. One of the exciting advancements of the field are living cells which can serve as molecular factories, diagnostics or therapeutics. A widely used chassis in synthetic biology is yeast due to simple and inexpensive culturing conditions and the ability to heterologously express eukaryotic proteins. In this thesis, we present work exploring and expanding biosensing and responding capabilities of engineered lab strain yeast.

Chapter 1 gives background information related to synthetic biology, living engineered biosensors, theranostics and more specifically on Saccharomyces cerevisiae general overview and applications in synthetic biology. Chapter 2 describes progress on establishing redox active peptides as a modular electrochemical interfacing language between electronics and engineered yeast. Chapter 3 covers yeast engineering as a heavy metal and metalloid biosensor, as well as the exploration of peptide-containing hydrobeads in conjunction with peptide-responsive yeast as a physical damage biosensor.

In Chapter 4, we establish living yeast biosensor for detection of pathogenic fungus Aspergillus fumigatus and expanded biosensing of other Aspergillus species, as well as additional optimization of the biosensing yeast’s signal-to-noise ratio, sensitivity and readout time. Chapter 5 demonstrates the utility of specific peptide proteases in combination with promiscuous GPCRs in living yeast biosensor for detection and differentiation of peptide variants differing in single amino acid. Lastly, in Chapter 6 we implement yeast sense-and-respond community which is activated by pheromone-secreting fungi and as a response secretes a toxin which kills sensed fungi.