Cell-free Transcription-translation Systems Research Articles

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.

Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.

Cell-free systems in the new age of synthetic biology

The advent of synthetic biology has ushered in new applications of cell-free transcription-translation systems. These cell-free systems are reconstituted using cellular proteins, and are amenable to modular control of their composition. Here, we discuss the historical advancement of cell-free systems, as well as their new applications in the rapid design of synthetic genetic circuits and components, directed evolution of biomolecules, diagnosis of infectious diseases, and synthesis of vaccines. Finally, we present our vision on the future direction of cell-free synthetic biology.

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

Cell-Free Expression Systems: From Gene Circuits to Self-Assembly Processes in a Test Tube

Cell-free transcription-translation (TX-TL) systems are becoming powerful platforms to construct complex biological systems in vitro through the expression of DNA programs. Considerable efforts have been made to improve those systems in the past decade. Our laboratory has developed the most efficient and versatile in vitro E. coli TX-TL system to express synthetic or natural DNA programs encoding for self-assembly processes. Either in test tube reactions, in emulsion droplets or in liposomes, cell-free TX-TL reactions are now used for research studies ranging from elementary gene circuits to minimal cells.I will present our custom-made cell-free TX-TL system, its current capabilities and limitations. Recently we have shown that entire phages can be synthesized in cell-free TX-TL reactions from their genomes. The bacteriophage T7, composed of about 60 genes, is entirely synthesized in a single test tube reaction from its 40 kbp genomic DNA. Replication of the T7 DNA instructions occurs concurrently with phage expression and self-assembly. The phage phiX174 composed of a dozen of genes can also be synthesized from its genome. Encapsulated inside cell-sized phospholipid liposomes, the TX-TL system is used to construct a prototype of minimal cell. I will present this cell-free synthetic biology platform, our last experiments and how this system can be used to study the relationship between information and self-organization.

RApid Parallel Protein EvaluatoR (RAPPER), from gene to enzyme function in one day.

Cell-free transcription-translation systems offer an effective and versatile platform to explore the impact of genetic variations on protein function. We have developed a protocol for preparing linear, mutagenic DNA templates for direct use in the PURE system, enabling the fast and semi-quantitative evaluation of amino acid variations on catalytic activity and stereo-selectivity in native and engineered variants of Old Yellow Enzyme.

A Quick in Vitro Pathway from Prokaryotic Genomic Libraries to Enzyme Discovery

Screening of prokaryotic genomes in order to identify enzymes with a desired catalytic activity can be performed in vivo in bacterial cells. We propose a strategy of in vitro expression screening of large prokaryotic genomic libraries based on Escherichia coli cell-free transcription-translation systems. Because cell-based expression may be limited by poor yield or protein misfolding, cell-free expression systems may be advantageous in permitting a more comprehensive screen under conditions optimized for the desired enzyme activity. However, monocistronic messages with an improved leader initiation context are typically used for protein production in vitro. Here, we describe successful use of a Pseudoalteromonas genomic DNA library for in vitro expression of DNA fragments carrying multiple open reading frames (ORFs) in the context of their authentic translation initiation sites and regulatory regions. We show that ORFs located far from the 5' and 3' ends of polycistronic transcripts can be expressed at a sufficient level in an in vitro transcription-translation system in order to allow functional screening. We demonstrate the overall cell-free functional screen strategy with the successful selection of an esterase from Pseudoalteromonas.

Characterization of Protein Expression and Folding in Cell-Free Systems by MALDI-TOF Mass Spectrometry

Recent advances in basic research, medicine, and biotechnology provide great motivation for the development of analytical tools to probe the behavior of target biomolecules in complex biological environments. Cell-free transcription-translation systems are an attractive medium for such studies, because they mimic several biochemical features of living cells, yet they are much more amenable to manipulation and spectroscopic analysis. However, few methods are currently available to characterize target proteins in cell-free systems. We have employed matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry for the detection and characterization of two cell-free expressed model proteins, cold shock protein A and apomyoglobin (apoMb) in cell-free systems. We exploited a combination of multiple selective isotope-labeling patterns for the identification of both full-length proteins and their in situ-generated proteolytic fragments. MALDI-TOF mass spectrometry-detected hydrogen/deuterium exchange, performed directly in the cell-free medium, allowed the assessment of apoMb's global degree of folding. The above methods are straightforward in that they do not require high levels of protein expression and allow the efficient characterization of both protein identity and global degree of folding.

High throughput cloning and expression strategies for protein production

Traditionally, the production of a recombinant protein requires a preliminary cloning step of the target gene into an expression vector before evaluating its cellular expression. Among current methods, site-specific recombination cloning techniques, which eliminate the use of restriction endonucleases and ligase, offer several advantages in the context of high throughput (HT) procedures. Rapid and highly efficient, the recombinational cloning technology is largely used for structural genomics and functional proteomics. However, the correct expression of some genes requires further optimization steps that are time-consuming and carried out at relatively late stages in the cloning-expression process. An alternative strategy is described where expression is tested in vitro before cloning the target gene. This technology, amenable to automation for HT studies, makes the expression of several hundreds of genes possible from PCR products in cell-free transcription–translation systems. Once this preliminary step is achieved, the PCR product, which gives satisfying expression levels, is selected, and then cloned in a plasmid for its cellular expression and perpetuation.

Cell-free production of biologically active polypeptides: application to the synthesis of antibacterial peptide cecropin.

An approach to preparative production of polypeptides, including uneasily testable, degradable, and toxic ones, is proposed on the basis of in vitro expression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The approach implies that a polypeptide of interest is synthesized as a fusion protein with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantitative monitoring of the polypeptide synthesis, as well as solubility and stability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated.

Continuous-flow cell-free translation, transcription-translation, and replication-translation systems.

The continuous inflow of consumable substrates and outflow of products tremendously increase the lifetime of cell-free protein synthesizing systems (1). As a consequence, the protein yield of the continuous-flow cell-free (CFCF) translation can be raised up to two orders of magnitude, as compared with classical batch systems. The CFCF protein synthesis technique can be utilized for basic research in the field of protein synthesis and folding as well as for numerous applications in biotechnology.

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80–82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli

The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity. After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70%. The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems. SLPI proved to be a potent inhibitor of translation in vitro. When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75%. The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA-metabolizing enzymes, such as RNase. In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61. Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin. On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA. Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPI/DNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E. coli. These results indicate that, in general, high-level expression of basic recombinant proteins in E. coli may be problematic.