Title:

Utilizing Plasmid DNA Hydrogels for Cell-Free Protein Production

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 low efficiency, batch-to-batch variability, and high costs during transcription and translation. In this research, we address these limitations by using plasmid DNA (pDNA) hydrogels as substrates for CFPS. pDNA is used to produce proteins and utilizing the reusability of pDNA hydrogels, enhances the efficiency and sustainability of CFPS processes. 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 attain DNA samples with concentrations up to 116 mg/mL and successfully purify plasmids. The CFPS process involves preparing cell extract via cell lysis, followed by adding nucleotides, RNA polymerase, and cofactors. pDNA encoding the desired protein is introduced, and the resulting protein is purified from the cell-free system. Future efforts will focus on purifying pUCBB-pT7-eGFP to optimize solution CFS with myTXTL T7 expression kit. We will then begin trials on chemically cross-linked pDNA hydrogels.

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