Protocols for Implementing an <em>Escherichia coli</em> Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Massachusetts Institute of Technology

Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro.

Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70.

A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink).

Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system.

Cell-free protein synthesis is becoming a serious alternative to cell-based protein expression. Cell-free systems can deliver large amounts of cytoplasmic recombinant proteins after a few hours of incubation. Recent studies have shown that membrane proteins can be also expressed in cell-free reactions and directly inserted into phospholipid membranes. In this work, we present a quantitative method to study in real time the concurrent cell-free expression and insertion of membrane proteins into phospholipid bilayers. The pore-forming protein Î±-hemolysin, fused to the reporter protein eGFP, was used as a model of membrane protein. Cell-free expression of the toxin in solution and inside large synthetic phospholipid vesicles was measured by fluorometry and fluorescence microscopy respectively. A quartz crystal microbalance with dissipation was used to characterize the interaction of the protein with a supported phospholipid bilayer. The cell-free reaction was directly incubated onto the bilayer inside the microbalance chamber while the frequency and the dissipation signals were monitored. The presence of pores in the phospholipid bilayer was confirmed by atomic force microscopy. A model is presented which describes the kinetics of adsorption of the expressed protein on the phospholipid bilayer. The combination of cell-free expression, fluorescence microscopy and quartz crystal microbalance-dissipation is a new quantitative approach to study the interaction of membrane proteins with phospholipid bilayers.

Î±-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression.

A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degradation machinery of E.Â coli. In dissecting the major reaction steps, we derive a coarse-grained enzymatic description of biosynthesis and degradation, from which ten relevant rate constants and concentrations are determined. Governed by zeroth-order degradation, protein expression follows a sharp transition from undetectable levels to constant-rate accumulation, without reaching steady state.

Coarse-grained dynamics of protein synthesis in a cell-free system.

Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes in vitro. Engineering synthetic gene circuits in a test tube, however, is seriously limited by the transcription repertoire of modern cell-free systems, composed of only a few bacteriophage regulatory elements. Here, we report the construction and the phenomenological characterization of synthetic gene circuits engineered with a cell-free expression toolbox that works with the seven E. coli sigma factors. The E. coli endogenous holoenzyme E(70) is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed with the six other sigma factors, two bacteriophage RNA polymerases, and a set of repressors. The circuit dynamics reveal the importance of the global mRNA turnover rate and of passive competition-induced transcriptional regulation. Cell-free reactions can be carried out over long periods of time with a small-scale dialysis reactor or in phospholipid vesicles, an artificial cell system. This toolbox is a unique platform to study complex transcription/translation-based biochemical systems in vitro.

An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells.

The physical interaction between the cytoskeleton and the cell membrane is essential in defining the morphology of living organisms. In this study, we use a synthetic approach to polymerize bacterial MreB filaments inside phospholipid vesicles. When the proteins MreB and MreC are expressed inside the liposomes, the MreB cytoskeleton structure develops at the inner membrane. Furthermore, when purified MreB is used inside the liposomes, MreB filaments form a 4-10 Î¼m rigid bundle structure and deform the lipid vesicles in physical contact with the vesicle inner membrane. These results indicate that the fibrillation of MreB filaments can take place either in close proximity of deformable lipid membrane or in the presence of associated protein. Our finding might be relevant for the self-assembly of cytoskeleton filaments toward the construction of artificial cell systems.

Assembly of MreB filaments on liposome membranes: a synthetic biology approach.

The synthesis of living entities in the laboratory is a standing challenge that calls for innovative approaches. Using a cell-free transcription-translation system as a molecular programming platform, we show that the bacteriophage T7, encoded by a 40 kbp DNA program composed of about 60 genes, can be entirely synthesized from its genomic DNA in a test tube reaction. More than a billion infectious bacteriophages T7 per milliliter of reaction are produced after a few hours of incubation. The replication of the genomic DNA occurs concurrently with phage gene expression, protein synthesis, and viral assembly. The demonstration that genome-sized viral DNA can be expressed in a test tube, recapitulating the entire chain of information processing including the replication of the DNA instructions, opens new possibilities to program and to study complex biochemical systems in vitro.

Jonghyeon Shin

Synthetic Biology Center,
Department of Bioengineering,
Synthetic Biology Center, Department of Bioengineering

Massachusetts Institute of Technology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Jonghyeon Shin

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Massachusetts Institute of Technology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Synthetic Biology Center,
Department of Bioengineering,
Synthetic Biology Center, Department of Bioengineering