Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene network

Pathways to cellular supremacy in biocomputing

Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the “genetic circuit” metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of “cellular supremacy” to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found.A.G.-M. was supported by the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1) and the European CSA on biological standardization BIOROBOOST (EU grant number 820699). T.E.G. was supported by a Royal Society University Research Fellowship (grant UF160357) and BrisSynBio, a BBSRC/ EPSRC Synthetic Biology Research Centre (grant BB/L01386X/1). P.Z. was supported by the EPSRC Portabolomics project (grant EP/N031962/1). P.C. was supported by SynBioChem, a BBSRC/EPSRC Centre for Synthetic Biology of Fine and Specialty Chemicals (grant BB/M017702/1) and the ShikiFactory100 project of the European Union’s Horizon 2020 research and innovation programme under grant agreement 814408

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.ISSN:1362-4962ISSN:0301-561

Design of Multipartite Transcription Factors for Multiplexed Logic Genome Integration Control in Mammalian Cells

Synthetic biology relies on rapid and efficient methods to stably integrate DNA payloads encoding for synthetic biological systems into the genome of living cells. The size of designed biological systems increases with their complexity, and novel methods are needed that enable efficient and simultaneous integration of multiple payloads into single cells. By assembling natural and synthetic protein-protein dimerization domains, we have engineered a set of multipartite transcription factors for driving heterologous target gene expression. With the distribution of single parts of multipartite transcription factors on piggyback transposon-based donor plasmids, we have created a logic genome integration control (LOGIC) system that allows for efficient one-step selection of stable mammalian cell lines with up to three plasmids. LOGIC significantly enhances the efficiency of multiplexed payload integration in mammalian cells compared to traditional cotransfection and may advance cell line engineering in synthetic biology and biotechnology.ISSN:2161-506

Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks.ISSN:1362-4962ISSN:0301-561

A designer cell-based histamine-specific human allergy profiler

Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients’ exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered within the allergy profiler rewires histamine input to the production of reporter protein, thereby integrating histamine levels in whole-blood samples with remarkable sensitivity and a wide dynamic range, allowing for rapid results or long-term storage of output, respectively. This approach provides non-intrusive allergy profiles for the personalized medicine era.ISSN:2041-172

CelloSelect – A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two β-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and β-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.ISSN:1096-7176ISSN:1096-718

Sensing and responding to allergic response cytokines through a genetically encoded circuit

While constantly rising, the prevalence of allergies is globally one of the highest among chronic diseases. Current treatments of allergic diseases include the application of anti-histamines, immunotherapy, steroids, and anti-immunoglobulin E (IgE) antibodies. Here we report mammalian cells engineered with a synthetic signaling cascade able to monitor extracellular pathophysiological levels of interleukin 4 and interleukin 13, two main cytokines orchestrating allergic inflammation. Upon activation of transgenic cells by these cytokines, designed ankyrin repeat protein (DARPin) E2_79, a non-immunogenic protein binding human IgE, is secreted in a precisely controlled and reversible manner. Using human whole blood cell culturing, we demonstrate that the mammalian dual T helper 2 cytokine sensor produces sufficient levels of DARPin E2_79 to dampen histamine release in allergic subjects exposed to allergens. Hence, therapeutic gene networks monitoring disease-associated cytokines coupled with in situ production, secretion and systemic delivery of immunomodulatory biologics may foster advances in the treatment of allergies.ISSN:2041-172