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Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Lookup NU author(s): Dr Siji Kavil, Alex Laverick, Dr Colette Whitfield, Dr Alice BanksORCiD, Dr Thomas Howard

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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Abstract

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Publication metadata

Author(s): Kavil S, Laverick A, Whitfield CJ, Banks AM, Howard TP

Publication type: Article

Publication status: Published

Journal: Journal of Visualized Experiments

Year: 2023

Volume: 196

Pages: e65500

Online publication date: 23/07/2023

Acceptance date: 22/05/2023

Date deposited: 10/08/2023

ISSN (electronic): 1940-087X

Publisher: Journal of Visualized Experiments

URL: https://doi.org/10.3791/65500

DOI: 10.3791/65500

Data Access Statement: Data supporting this publication are openly available at: 10.25405/data.ncl.22232452

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