Honors Theses and Capstones

Date of Award

Spring 2024

Project Type

Senior Honors Thesis

College or School

COLSA

Department

Chemistry Department & the Biochemistry, Molecular, & Cellular Biology Department

Program or Major

Biochemistry

Degree Name

Bachelor of Science

First Advisor

Dr. Nathan J. Oldenhuis

Abstract

Proteins play an important role in various biological processes and are crucial components in biomedical research and therapeutics. Cell-free protein synthesis (CFPS) has provided a valuable approach for rapid protein production outside living cells, involving both transcription and translation processes. However, traditional CFPS methods encounter challenges such as high cost, relatively low yields, and scalability. The end goal of our project is to incorporate our template into the hydrogel. In this work, we report advancements towards that by trying to optimize our solution of CFPS. Unfortunately, the standards for pDNA hydrogel synthesis are not met by the pDNA production and purification methods suitable for an academic lab. To meet the demand for large amounts of pDNA, a scalable and standardized method applicable across academic labs is necessary. We achieve this by adapting large-scale industrial protocols to academic environments, enabling mass production of pDNA. Through this approach, we have produced pure DNA samples with concentrations up to 116 mg/mL. Before attempting cell-free synthesis of chemically cross-linked pDNA hydrogels, we began with solution CFPS using the commercially available myTXTLSigma 70 Master Mix kit. Initial trials have only been successful with the positive control plasmid provided with the kits. Future work will focus on optimizing solution CFPS before moving onto chemically cross-linked hydrogels.

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