RESUMEN
Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.
Asunto(s)
Extractos Celulares/química , Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa Pancreática/metabolismo , Biología Sintética/métodos , Animales , Bovinos , Sistema Libre de Células/metabolismo , Codón/genética , Enzimas Inmovilizadas/metabolismo , Escherichia coli/genética , Biosíntesis de Proteínas , ARN de Transferencia/análisisRESUMEN
Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.
Asunto(s)
Técnicas Biosensibles , Disruptores Endocrinos/análisis , Proteínas Recombinantes de Fusión/síntesis química , Receptores beta de Hormona Tiroidea/química , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Proteínas Recombinantes de Fusión/químicaRESUMEN
Cell-free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell-free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell-free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell-free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell-free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems' protein synthesis capabilities. Lyophilization provides the additional benefit of long-term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely "cell-free" systems.
Asunto(s)
Biotecnología/métodos , Sistema Libre de Células/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Recombinantes/metabolismo , Escherichia coli/metabolismo , Filtración , Liofilización , MuramidasaRESUMEN
The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.