Methods for embedding cell-free protein synthesis reactions in macro-scale hydrogels
Author(s)
Kavil, Siji
Laverick, Alex
Whitfield, Colette J
Banks, Alice M
Howard, Thomas P
Type
Journal Article
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.
Date Issued
2023-06
Date Acceptance
2023-06-01
Citation
Journal of Visualized Experiments, 2023, (196), pp.1-14
ISSN
1940-087X
Publisher
MyJove Corporation
Start Page
1
End Page
14
Journal / Book Title
Journal of Visualized Experiments
Issue
196
Copyright Statement
Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial 3.0 License
Identifier
https://app.jove.com/t/65500/methods-for-embedding-cell-free-protein-synthesis-reactions-macro
Publication Status
Published
Article Number
e65500
Date Publish Online
2023-06-23