Cell-free synthetic biology: thinking outside the cell

Abstract

Cell-free synthetic biology is emerging as a powerful approach aimed to understand, harness, and expand the capabilities of natural biological systems without using intact cells. Cell-free systems bypass cell walls and remove genetic regulation to enable direct access to the inner workings of the cell. The unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the rapid development of engineering foundations for cell-free systems in recent years. These efforts have led to programmed circuits, spatially organized pathways, co-activated catalytic ensembles, rational optimization of synthetic multi-enzyme pathways, and linear scalability from the micro-liter to the 100-liter scale. It is now clear that cell-free systems offer a versatile test-bed for understanding why nature's designs work the way they do and also for enabling biosynthetic routes to novel chemicals, sustainable fuels, and new classes of tunable materials. While challenges remain, the emergence of cell-free systems is poised to open the way to novel products that until now have been impractical, if not impossible, to produce by other means.

Figure 1

(a) Synthesis platforms in synthetic biology. While the majority of synthetic biology projects are performed in vivo, in vitro systems are emerging as a complementary technology. In vitro systems can be further subdivided into crude extract cell-free systems (CECFs), and synthetic enzymatic pathways (SEPs). The flow diagram overviews how the different systems are related. (b) Cell-free platforms (listed in bold) can be further grouped to reflect their applications, which include translation systems and small molecule production. The strength of the arrows loosely represents the relative amount of published papers to date, but does not represent the platform’s ability and/or application potential.

Figure 2

Milestones in the scalability of batch E. coli extract cell-free protein synthesis reactions. Modern batch cell-free reactions were debuted by Kim et al. (1996) at the 20 µL scale with yields of 0.1 g L⁻¹ chloramphenicol acetyltransferase (CAT). Over the last 15 years batch E. coli extract cell-free protein synthesis reactions have increased in reaction yields and scale to 60 µL reaction volume and 0.15 g L⁻¹ CAT (Kim and Swartz, 1999), 100 µL reaction volume and 0.23 g L⁻¹ CAT (Kim and Swartz, 2001), 500 µL reaction volume and 0.56 g L⁻¹ CAT (Voloshin and Swartz, 2005), 2 mL reaction volume and 1.2 g L⁻¹ CAT (Jewett et al., 2008), and most recently 100 L reaction volume and 0.7 g L⁻¹ recombinant human granulocyte macrophage colony-stimulating factor in 2011, creating the first ever industrially relevant cell-free protein synthesis reaction (Zawada et al., 2011).

Figure 3

Flow-diagram of typical CECF reaction. Briefly, a strain is chosen or engineered as appropriate and cells are grown and harvested; the culture is lysed, buffer is exchanged through desalting, and the extract is then normally subject to pre-incubation for translation reactions; lastly, the crude-extract is incubated under typical reaction conditions and the product is assayed or recovered. Blue boxes represent cellular growth and harvest, green boxes denote preparation of the cell-free extract, and red boxes denote cell-free product synthesis and recovery.

Figure 4

Programming cell-free circuits. (a) A general schematic of a combined transcription and translation reaction cascade. Here a T7 bacteriophage RNA polymerase (T7 RNAP) is used to prime the reaction cascade by binding to its promoter region and transcribing a secondary bacteriophage RNAP (SP6). This in turn selectively binds the SP6 promoter region prefacing the expression of the protein of interest (luciferase). (b) A two-switch DNA oscillator is controlled by the addition of two activator sequences (A1 DNA and A2 DNA), which can bind to and activate a respective DNA switch, an inhibitor sequence (I1 DNA), and two enzymes RNA polymerase and RNase H. DNA Switch 1 is turned ON when A1 DNA binds to it, thus completing the RNA polymerase promoter region. Subsequently, DNA Switch 1 undergoes transcription to produce A2 RNA complementary to A2 DNA. The A2 RNA is able to initiate binding to release the activator DNA sequence A2 from the DNA Switch 2, thus turning DNA Switch 2 OFF. The A2 RNA of the A2 RNA/DNA hybrid is then digested by RNase H and releases the activator A2 DNA. DNA Switch 2 is turned ON when A2 DNA binds and subsequently undergoes transcription to produce the I1 RNA sequence complementary to I1 DNA. Binding of I1 DNA by I1 RNA releases A1 DNA thus allowing DNA Switch 2 to be turned ON. Once again, the I1 RNA of the I1 RNA/DNA hybrid is digested by RNase H and releases the DNA inhibitory sequence I1, thus turning DNA Switch 1 OFF. The process is repeated and the oscillatory behavior is sustained over time. c) The cis-repressed (CR) riboswitch is designed with a complementary sequence able to bind the ribosome binding site (RBS) of the protein of interest and inhibit translation. Expression of a second regulatory RNA deemed the trans-activating RNA (taRNA) can bind to and release the CR region from the RBS and activate translation.

Figure 5

Generalized in vitro compartmentalization (IVC) example for directed enzyme evolution. Initially, a gene library of an enzyme of interest is constructed and attached to a “substrate” in order to establish a physical link between the genotype and phenotype. For example, the substrate could be composed of a microbead displaying small molecules that interacts with the downstream enzyme of interest. The gene library is compartmentalized in a water-in-oil emulsion with on average less than one gene per water droplet where it undergoes combined transcription and translation (step 1). If successful, the synthesized enzyme will interact with the substrate to make a product (step 2). IVC prevents the synthesized protein from interacting with substrates in other compartments. The emulsion is broken (step 3) and the winning products can be recovered along with the gene (step 4), through affinity purification for example. After capture, variants can be amplified and additional cycles of selection and amplification can be carried out (step 5). For specific examples of IVC directed evolution designs see Agresti et al., 2005; Bernath et al., 2004; Doi et al., 2004; Griffiths and Tawfik, 2003; Levy and Ellington, 2008; Levy et al., 2005; and Tawfik and Griffiths, 1998), and for detailed protocols see Davidson et al., 2001; or Miller et al., 2006.

Figure 6

Spatial organization techniques include (a) mRNA display, (b) protein scaffolding, (c) CLEA particles, and (d) foam dispersion.