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

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

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.

Synthetic biology integrates diverse engineering disciplines to design and engineer biologically based parts, devices, and systems that can perform functions that are not found in nature. Most synthetic biology approaches are still bound to living cells. By contrast, cell-free synthetic biology systems facilitate unprecedented levels of control and freedom in design, enabling increased flexibility and a shortened time for engineering biological systems while eliminating many of the constraints of traditional cell-based gene expression methods^1,2,3. CFPS is being used in an in....

1. Cell lysate buffer and media preparation

1. Preparation of 2x YT+P agar and medium
   1. Prepare 2x YT+P agar by measuring out 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 40 mL/L 1 M K₂HPO₄, 22 mL/L 1 M KH₂PO₄, and 15 g/L agar. For the 2x YT+P broth, follow the previous composition but omit the agar.
   2. Sterilize by autoclaving the 2x YT+P.
2. Preparation of the S30A buffer
   1. Prepare the S30A buffe.......

This protocol details two methods for embedding CFPS reactions into hydrogel matrices, with Figure 1 presenting a schematic overview of the two approaches. Both methods are amenable to freeze-drying and long-term storage. Method A is the most utilized methodology for two reasons. First, it has been shown to be the most applicable method for working with a range of hydrogel materials¹¹. Second, Method A allows for the parallel testing of genetic constructs. Method.......

Outlined here are two protocols for the incorporation of E. coli cell lysate-based CFPS reactions into agarose hydrogels. These methods allow for simultaneous gene expression throughout the material. The protocol can be adapted for other CFPS systems and has been successfully conducted with commercially available CFPS kits in addition to the laboratory-prepared cell lysates detailed here. Importantly, the protocol allows for gene expression in the absence of an external liquid phase. This means that the system i.......

The authors greatly acknowledge the support of the Biotechnology and Biological Sciences Research Council awards BB/V017551/1 (S.K., T.P.H.) and BB/W01095X/1 (A.L., T.P.H.), and the Engineering and Physical Sciences Research Council - Defence Science and Technology Laboratories award EP/N026683/1 (C.J.W., A.M.B., T.P.H.). Data supporting this publication are openly available at: 10.25405/data.ncl.22232452. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising.