By 2030, up to half of the world’s population is projected to suffer from water insecurity: a chronic scarcity of potable water due to rapidly warming temperatures, increased agricultural demand, and pollution. The health impacts of contaminated water are profound: hundreds of thousands of global deaths each year are ascribed to the consumption of untreated water containing fecal bacteria, heavy metals, or industrial runoff. Although analytical chemistry enables ultrasensitive detection of these water contaminants, most such strategies rely on highly centralized equipment that is too expensive and unwieldy for distributed water monitoring, especially in low-resource settings. Synthetic biology offers an appealing alternative to this paradigm, since microbial mechanisms for detecting contaminants can be easily re-engineered to output visible signals that are interpretable without sophisticated electronics. In particular, cell-free biosensors made from bacterial lysates could be fieldable diagnostic tools because they are safer, cheaper, and more shelf-stable than living cells. In this dissertation, I describe a series of innovations that enable cell-free gene expression to be a compatible platform for molecular diagnostics. I delineate the aspects of the extract preparation process that most impact the function of cell-free genetic regulators. Using an optimized, streamlined protocol, I highlight strategies for developing a core set of molecular sensors against key water contaminants. Then, I adapt cascaded genetic circuitry to sensitize and amplify the response function of these biosensors, allowing them to be used for practical diagnostics, highlighting the detection of lead in tap water as an important case study. I describe a handful of further projects to develop cell-free sensors for practical utility and also outline logical next steps for this platform, including evolution and screening strategies for rapidly developing new artificial molecular sensors and proof-of-concept experiments for population-scale water monitoring. Taken as a whole, this work details several innovations that may be transformative for improving global public health in years to come.

Creator

• Silverman, Adam Daniel

DOI

• https://doi.org/10.21985/n2-4y06-ab42

Subject

• Bioengineering
• Chemical engineering
• Public health

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15551
• etdadmin_upload_817796

Keyword

• diagnostics
• water quality
• cell-free
• synthetic biology

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright