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Biology Beyond the Cell: the How and Why of Cell-Free Systems

Light outside shining in a cell.

The Cell Membrane: A Blessing and a Curse

The cell membrane envelops a cytosol teeming with organelles, unstable molecules, enzymes, and genetic information – everything required for metabolism and replication. But it also keeps out anything that would destabilize the delicate, dynamic equilibrium of life.

While the membrane is essential for the cell, it is often also an inconvenience for biological engineers. Preparing cells to accept foreign DNA, getting the DNA across the cell membrane, and integrating the new instruction set into the organism’s genome are time-consuming and laborious tasks. And researchers are increasingly interested in expanding our repertoire of host organisms to more exotic species, which lack streamlined transformation protocols.

Most of the processes of protein production and metabolism don’t have any fundamental requirement for encapsulation. If no cell is required for your application, then why bother?

Cell-free expression systems, which take expression machinery out of the cell and into a test tube, are a solution to this seemingly intractable problem.

These systems can be used to answer fundamental biological questions, such as studying expression in “protocells”. They are equally useful for quickly prototyping genetic and metabolic systems: for example, rapidly optimizing metabolic pathways to maximize product titers. They can even be used as on-demand biosensing and biomanufacturing platforms. If you’re interested in learning more about what cell-free systems are, why they’re useful, and how you can get started, read on!

Putting It All (Back) Together

What components are required for translation to occur outside of the cell? All cell-free protein expression systems contain at least these three parts.

  1. Translational machinery. This includes ribosomes and tRNAs, as well as initiation, elongation, and release factors required for translation, and aminoacyl tRNA synthetases to charge tRNAs with their cognate amino acids.
  2. Energy. GTP and ATP to power translational elongation and tRNA charging. Sugars, phosphorylated glycolytic intermediates, or other phosphorylated compounds are used as energy sources to make sure that high concentrations of nucleotide triphosphates are maintained over the course of the reaction, which can last up to several hours.
  3. A messenger RNA to be translated. This can be produced outside of the cell-free reaction via in vitro T7 transcription, or within the cell-free reaction by adding a DNA template and NTPs and using either T7 RNA polymerase or a native polymerase present in the cell lysate.

Cell-free systems recapitulate translation, and often transcription and central metabolism, outside of the cell. However, it’s important to remember the three main differences between cell-free and cell-based systems.

  1. Compartmentalisation and spatial organization. Cell-free expression systems lack a barrier between a biochemical reaction system and the surrounding environment, as well as barriers between functionally distinct compartments. All biochemical reactions in cell-free systems are taking place in a homogenous environment.
  2. Dilution. Cell-free systems are an order of magnitude more dilute, in terms of their macromolecular content, than cells. Not only are the concentrations of gene expression machinery lower, but the degree of macromolecular crowding is lower as well, which can influence biochemical reaction rates and equilibria.
  3. A genome. Chromosomal DNA is digested or purified out of cell-free systems. Because cell-free systems lack a genome to program behavior, the only instructions that the reaction will carry out are those that you provide.

Why Should You Go Cell-Free?

Because cell-free systems lack a cell membrane, they have several advantages over cellular expression.

  • Faster – There are no long days of transformation and cell growth in a cell-free experiment. A typical cell-free reaction takes hours to complete, not days. The difference in speed between in-cell and cell-free becomes even greater when working in organisms with complex or unoptimized transformation protocols, or longer culture times. Cell-free can be a great way to prototype genetic parts for novel host organisms, genetic circuits, and even entire metabolic pathways without having to integrate any genetic information into an intractable host.
  • Biochemically flexible – The intracellular concentrations of small molecules, metabolites, and enzymes are all highly regulated. It can be difficult to tune their concentrations for optimal yield or genetic system performance, or even to monitor what those concentrations are. You have much more control over the biomolecular composition in a cell-free system.  You can add novel cosolutes, or even use lipid micelles to re-encapsulate gene expression machinery, to investigate how these modifications impact gene expression.
  • Open – The openness of cell-free systems is helpful when modifying system chemistry, but it is especially useful for applications that require communication with an outside environment, such as sensing. It is currently difficult to engineer cellular sensing platforms that respond to molecules that can’t cross the cell membrane, like nucleic acids. In a cell-free sensing system, that barrier doesn’t exist, allowing for simple detection of molecules like viral RNA.

…and Why Should You Not?

The differences between the cellular and cell-free environments can lead to differences in the experimental approach between the two expression platforms.

  • Not (yet) high-throughput – High-throughput flow-cytometry and NGS-based genetic characterization experiments are well-established for cellular platforms, which conveniently package genetic instructions together with their output. Tens of thousands of genetic variants can be screened for function using flow cytometry and/or NGS. The number of variants that can be screened in a traditional single cell-free experiment is several orders of magnitude less than in high-throughput in vivo assays.
  •  In vivo does not (necessarily) equal in vitro – Be aware that cell-free results do not necessarily directly map to in vivo system performance, or even to other cell-free systems. Because of differences in the concentrations of system components, cell-free output between systems tends to be comparable, but not identical.
  • It’s not economical for large scale protein preparations. While yields of up to 2.3 g/L of protein have been reported for cell-free systems, which makes it competitive with cell-based expression, you would need a culture volume of ~1000x the volume of the cell-free reaction volume. This means it is not an economical system for creating large quantities of recombinant protein.

Choosing a System

Prokaryotic or eukaryotic, homebrewed or commercial, crude extract-based or reconstituted? The choice of system depends on your specific needs and constraints.

Which species? Unless you are prototyping genetic parts in, or need the expression machinery of a specific organism, an E. coli-based cell-free system should be your first choice. The oldest and most widely-used family of cell-free systems, E. coli systems have been optimized for reliable gene expression and high-yield protein production.

Going commercial? Producing your own cell extract requires equipment and expertise to culture and lyse large volumes of cells. A handful of suppliers sell their own cell-free systems, derived from model organisms and workhorse cell lines, but they’re not cheap. Expect to pay at least $7 per reaction, versus cents per reaction for a homebrewed system.

Crude or reconstituted? Most cell-free expression systems are made with crude cell lysate, which contains many more enzymes than the core gene expression machinery. This can be a good thing—modern E. coli lysate-based systems contain chaperones and most of central metabolism, which increases protein yields and product titers. If you need to completely remove the activity of a certain enzyme, a reconstituted system known as PURE (Protein synthesis Using Recombinant Elements) may be the right option. In PURE, each protein required for gene expression is purified and added to the reaction, giving you maximum control over the reaction composition. Streamlined protocols have recently been published to economically produce both types of systems, but you can also purchase a kit.

To Sum It up

Cell-free systems are unencapsulated biosynthetic systems which are useful for rapid genetic system development and bioproduct synthesis. If you’re looking to speed up the development of a new biosynthetic pathway, investigate a biochemical process in a simpler system, or would like a simpler way to use the expression machinery from a genetically intractable host, going cell-free might be right for you.

Further Reading

Dudley QM, Karim AS & Jewett MC. Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell. Biotechnol J. (2015) 10(1): 69–82. DOI: 10.1002/biot.201400330

Garamella J, Marshall R, Rustad M & Noireaux V. The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synthetic Biology (2016) 5(4):344-355. DOI: 10.1021/acssynbio.5b00296

Thermo Fisher Scientific. Overview of Protein Expression. Protein Biology Resource Library.

Shimizu Y, Kanamori T, Ueda T. Protein synthesis by pure translation systems. Methods. (2005) 36(3):299-304. DOI: 10.1016/j.ymeth.2005.04.006

Shoba. An Intro to Cell-free Protein synthesis. Bitesize Bio. 2 March 2009.

Shoab. Solved: Low Yields in Cell-Free Protein Synthesis. Bitesize Bio. 21 April 2009.

Light outside shining in a cell.

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