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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: June 23rd, 2023

DOI:

10.3791/65500

1School of Natural and Environmental Sciences, Newcastle University, 2Department of Life Sciences, Imperial College London
* These authors contributed equally

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 methods1,2,3. CFPS is being used in an in....

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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.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
Material
3-PGA Santa Cruz Biotechnology sc-214793B
Acetic Acid Sigma-Aldrich A6283
Agar Thermo Fisher Scientific A10752.22
Agarose Severn Biotech 30-15-50
Amino Acid Sampler Kit VWR BTRABR1401801
ATP Sigma-Aldrich A8937-1G
cAMP Sigma-Aldrich A9501-1G
Coenzyme A (CoA) Sigma-Aldrich C4282-100MG
CTP Alfa Aesar J14121.MC
DTT Thermo Fisher Scientific R0862
Folinic Acid Sigma-Aldrich F7878-100MG
GTP Carbosynth NG01208
HEPES Sigma-Aldrich H4034-25G
K-glutamate Sigma-Aldrich G1149-100G
Lysozyme Sigma-Aldrich L6876-1G
Mg-glutamate Sigma-Aldrich 49605-250G
NAD Sigma-Aldrich N6522-250MG
PEG-8000 Promega V3011
Potassium Hydroxide (KOH) Sigma-Aldrich 757551-5G
Potassium Phosphate Dibasic (K2HPO4) Sigma-Aldrich P3786-500G
Potassium Phosphate Monobasic (KH2PO4) Sigma-Aldrich RDD037-500G
Protease Inhibitor cocktail Sigma-Aldrich P2714-1BTL
Qubit Protein concentration kit Thermo Fisher Scientific A50668
Rossetta 2 DE 3 E.coli Sigma-Aldrich 71397-3
Sodium Chloride (NaCl) Sigma-Aldrich S9888-500G
Spermidine Sigma-Aldrich 85558-1G
Tryptone Thermo Fisher Scientific 211705
Tris Sigma-Aldrich GE17-1321-01
tRNA Sigma-Aldrich 10109541001
UTP Alfa Aesar J23160.MC
Yeast Extract Sigma-Aldrich Y1625-1KG
Equipment
1.5 mL microcentrifuge tubes Sigma-Aldrich HS4323-500EA
10K MWCO dialysis cassettes Thermo Fisher Scientific 66381
15 mL centrifuge tube Sarstedt 62.554.502
50 mL centrifuge bottles Sarstedt 62.547.254
500 mL centrifuge bottles Thermo Fisher Scientific 3120-9500
Alpha 1-2 LD Plus freeze-dryer Christ part no. 101521, 101522, 101527
Benchtop Centrifuge Thermo Fisher Scientific H-X3R
Black 384 well microtitre plates Fischer Scientific 66
Cuvettes Thermo Fisher Scientific 222S
Elga Purelab Chorus Elga #####
Eppendorf Microcentrifuge 5425R Eppendorf EP00532
High Speed Centrifuge Beckman Coulter B34183
JMP license SAS Institute 15
Magnetic Stirrer Fischer Scientific 15353518
Parafilm Amcor PM-966
Photospectrometer (Biophotometer) Eppendorf 16713
Pipettes and tips Gilson #####
Precision Balance Sartorius 16384738
Qubit 2.0 Fluorometer Thermo Fisher Scientific Q32866
Shaking Incubator Thermo Fisher Scientific SHKE8000
Sonic Dismembrator (Sonicator) Thermo Fisher Scientific 12893543
Static Incubator Sanyo MIR-162
Syringe and needles Thermo Fisher Scientific 66490
Thermo max Q8000 (Shaking Incubator) Thermo Fisher Scientific SHKE8000
Varioskan Lux platereader Thermo Fisher Scientific VLBL00GD1
Vortex Genie 2 Cole-parmer OU-04724-05
VWR PHenomenal pH 1100 L, ph/mv/°c meter VWR 662-1657

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