Cell-Free Synthetic Biology: Engineering Beyond the Cell

Abstract

Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biology as well as a protein production technology in years to come.

Figure 1.

Cell-free protein synthesis (CFPS) platforms allow for increased flexibility and shortened process timelines to create a variety of high-value recombinant proteins. This technology provides platforms from numerous organisms with varied complexity to meet the needs of the specific target proteins. CFPS also decouples catalyst synthesis and catalyst usage, traditionally interconnected during in vivo recombinant protein production schemes. For CFPS, catalyst synthesis involves cell growth, cell lysis, and extract processing that removes genomic DNA to create crude extract. The crude extract may be frozen for future use or used directly for catalyst usage. This process requires NTPs, DNA, amino acids, and an energy source to produce various proteins with applications in the synthetic biology field.

Figure 2.

Assembly of macromolecules in cell-free protein synthesis (CFPS) reactions. (A) Complete in vitro assembly of adenosine triphosphate (ATP) ATP synthase with hypothesized genetic regulation. The ATP operon is added to a crude Escherichia coli extract and transcribed into a single messenger RNA (mRNA). Proteins are expressed at various levels determined by operon regulation. Matthies et al. (2011) suggest synthesis of correctly assembled ATP synthase complexes is dependent on specific expression levels of the subunits, correlated to the subunit stoichiometry in the complex. It is hypothesized that intermediate assemblies may also activate the expression of other subunits in the operon, allowing for sequential assembly processes (Kucharczyk et al. 2009). Thus, combined expression and assembly in CFPS systems allows for an additional level of assembly complexity for analysis. (B) In vitro integrated synthesis, assembly, and translation (iSAT) method of constructing ribosomes enables synthesis of active firefly luciferase in a one-pot reaction. iSAT enables one-step coactivation of ribosomal RNA (rRNA) transcription, assembly of ribosomal subunits, and synthesis of active protein by these ribosomes in same compartment. This process begins with T7 RNAP polymerase transcribing rRNA and luciferase mRNA. Ribosomal subunits are reconstituted from mature rRNA and ribosomal components previously purified or synthesized in vitro. Newly assembled ribosomes translate mRNA encoding the reporter protein luciferase to assess its activity.

Figure 3.

Schematic representation of cotranslational incorporation of a nonstandard amino acid (nsAA) using an orthogonal translation system and amber suppression. The orthogonal aminoacyl-tRNA synthetase (o-aaRS) first binds its cognate nsAA and cognate o-tRNA. The o-aaRS then catalyzes the aminoacylation of the o-tRNA. The aminoacyl-tRNA (aa-tRNA) is then released from the o-aaRS and transported to the ribosome by the elongation factor-Tu (EF-Tu). The aa-tRNA associates with the A-site of the ribosome and its anticodon binds the complementary triplet codon of the messenger RNA (mRNA). The ribosome then ligates the nsAA to the growing peptide chain. When release factor 1 (RF-1) outcompetes the aa-tRNA for binding at the UAG amber stop codon, the protein is truncated, which results in a decrease of nsAA incorporation efficiency. This problem has been overcome by recoding all TAG codons to the synonymous TAA codon, permitting the deletion of RF-1 (Johnson et al. 2011; Loscha et al. 2012b; Ohtake et al. 2012; Lajoie et al. 2013).

Figure 4.

Glycoprotein production in cell-free systems. The Campylobacter jejuni N-glycan biosynthesis pathway, which contains glycosyltransferases and a flippase, is expressed in vivo in Escherichia coli. These enzymes assemble sugar monomers (bacillosamine, N-acetylglucosamine, glucose) onto a lipid anchor (undecaprenyl pyrophosphate) in the membrane to form lipid-linked oligosaccharides (LLOs) (top). Similarly, the C. jejuni oligosaccharyltransferase (OST), PglB, is expressed in vivo in E. coli (bottom). LLOs and PglB are purified and added to the in vitro glycoprotein synthesis reaction. Addition of purified LLOs and PglB to the cell-free protein synthesis (CFPS) reaction results in the synthesis of fully glycosylated AcrA, a C. jejuni glycoprotein (Guarino and DeLisa 2012).

Figure 5.

In vitro prototyping of genetic networks. (A) Overview of in vitro prototyping for speeding up in vivo design–build–test cycles. In vitro prototyping allows for a genetic part or network to be quickly screened for specific characteristics in vitro before implementation in vivo. First, in the “Design” stage, the genetic parts or networks are designed, informed by computational models or literature. Next, in the “Build” stage, several designs are built. In the “Test” phase, built designs are assayed in vitro. If the Test stage does not yield the desired behavior, one reinitiates the cycle n number of times until the desired characteristic is achieved. The Test stage can also inform the modeling and allow for better models for the Design stage. Once the desired characteristics are found, top candidates that behave as expected can be implemented in vivo, with an increased likeliness of being functional. (B) Variables for characterization and optimization. In vitro prototyping can occur by characterizing or optimizing various levels on the genetic network “abstraction hierarchy.” At the most basic level, transcriptional and translational parts or purified components of a network can be analyzed for functionality. In vitro prototyping can also be applied at the device level to assess input–output relationships. Finally, an in vitro systems level analysis allows for an isolated study of how multiple genetic devices feed into each other and a preview of the overall network behavior.