Design and Implementation of a Glucose-Responsive Genetic Switch Circuit in Cell-Free Systems for the Management of Diabetes

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Cell-free protein synthesis (CFPS) systems are a widely used research tool in systems and synthetic biology and a promising platform for manufacturing of proteins and chemicals. Advances in cell-free extract preparation and energy regeneration mechanisms have evolved the platform from merely an investigative tool for understanding fundamental biochemical processes to a promising technology for just-in-time manufacturing of therapeutically important biologics and high-value small molecules. The CFPS reaction mixtures, typically composed of the proteins and DNA templates to enable transcription, translation, and gene regulation, can be lyophilized and stored at room temperature for up to a year, and they can then be reconstituted to synthesize proteins on demand, enabling their use as portable expression systems. The aim of this work is to advance the platform further, and engineer it for use in an important therapeutic application: management of diabetes. In 2021, approximately 537 million adults were living with diabetes and 6.7 million people died due to diabetes-related deaths; by 2045 the total cases is expected to rise to 783 million. Over 75% of diabetic adults live in low and middle income countries where the distribution of current formulations of therapeutic hormones (e.g., insulin) are challenging due to high cost associated with cold-chain requirements. These alarming facts underscore the need for rapid, effective, and affordable tools and therapies for diabetes management. We believe that CFPS platforms can be effectively utilized to address this disease. Toward this need, we have developed a novel input-dependent protein synthesis strategy: a cell-free genetic switch circuit that senses glucose levels (e.g., in the human blood) and drives the synthesis of either protein A or B or a combination of both depending on the glucose level. Due to the modular nature of the engineered gene expression cassettes, proteins A and B can be replaced with hormones such as Insulin or Glucagon, enabling a personalized hormone dose to be synthesized on-demand. This body of work starts by diving into the history and evolution of cell-free biology, and its current implementations and limitations. We also highlight the need for mathematical modeling, in particular, mechanistic biophysical models of protein synthesis and integrated metabolic models that couple metabolism with gene expression in order to further improve the CFPS platform. Toward facilitating the validation of computational metabolic models, this work focuses next on the development of a high-throughput liquid chromatography-mass spectrometry approach for accurately quantifying key cellular metabolites. Then this work describes the development of biophysically-motivated models for simulating transcription and translation dynamics in CFPS systems, with the potential to model in vivo systems due to the modular nature of the equations. This work focuses next on the construction and analysis of a gluconate-responsive biosensor in a commercially-available reconstituted CFPS, PURExpress. Finally, after laying the required foundations for systematic and effective design and analysis of the CFPS system, this work focuses on the big picture of the PhD work: development of a glucose-responsive switch circuit for the management of diabetes.

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225 pages


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Cell free systems; Diabetes; Genetic circuit; Glucose sensing; Mechanistic Modeling; Synthetic Biology


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Union Local


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Varner, Jeffrey

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DeLisa, Matthew
Helmann, John

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Chemical Engineering

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Ph. D., Chemical Engineering

Degree Level

Doctor of Philosophy

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Government Document




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Attribution-NonCommercial-ShareAlike 4.0 International


dissertation or thesis

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