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ACS Synthetic Biology

Pimkhuan Hannanta-Anan, Brian Y Chow
As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated hetero-dimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium...
May 24, 2018: ACS Synthetic Biology
Pichet Praveschotinunt, Noemie-Manuelle Dorval Courchesne, Ilona den Hartog, Chaochen Lu, Jessica J Kim, Peter Q Nguyen, Neel S Joshi
The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of tracking microbes in vivo within the gastrointestinal tract by programming them to incorporate non-standard amino acids (NSAA) and labeling them via click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered Escherichia coli to incorporate p-azido-L-phenylalanine (pAzF) in place of the UAG (amber) stop codon...
May 23, 2018: ACS Synthetic Biology
Yan Chen, Yanmin Wan, Nanxi Wang, Zhe Yuan, Wei Niu, Qingsheng Li, Jiantao Guo
Large efforts have been devoted to the genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells...
May 22, 2018: ACS Synthetic Biology
Ana M Mariscal, Shigeyuki Kakizawa, Jonathan Y Hsu, Kazuki Tanaka, Luis González-González, Alicia Broto, Enrique Querol, Maria Lluch-Senar, Carlos Piñero-Lambea, Lijie Sun, Philip D Weyman, Kim S Wise, Chuck Merryman, Gavin Tse, Adam J Moore, Clyde A Hutchison, Hamilton O Smith, Masaru Tomita, J Craig Venter, John I Glass, Jaume Piñol, Yo Suzuki
Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies...
May 22, 2018: ACS Synthetic Biology
Göksel Mısırlı, Tramy Nguyen, James Alastair McLaughlin, Prashant Vaidyanathan, Timothy Jones, Douglas Densmore, Chris J Myers, Anil Wipat
Computational models are essential to engineer predictable biological systems and to scale up this process for complex systems. Computational modelling often requires expert knowledge and data to build models. Clearly, manual creation of models is not scalable for large designs. Despite several automated model construction approaches, computational methodologies to bridge knowledge in design repositories and the process of creating computational models has still not been established. This paper describes a workflow for automatic generation of computational models of genetic circuits from data stored in design repositories using existing standards...
May 21, 2018: ACS Synthetic Biology
Jeffrey C Moore, Agustina Rodriguez-Granillo, Alejandro Crespo, Sridhar Govindarajan, Mark Welch, Kaori Hiraga, Katrina Lexa, Nicholas Marshall, Matthew David Truppo
Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes...
May 21, 2018: ACS Synthetic Biology
Jared Weaver, Steven G Boxer
Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded non-canonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Towards that goal, we developed a method to transfer amber codon suppression machinery developed for E...
May 15, 2018: ACS Synthetic Biology
Oren Yishai, Madeleine Bouzon, Volker Döring, Arren Bar-Even
Assimilation of one-carbon compounds presents a key biochemical challenge, which limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2...
May 15, 2018: ACS Synthetic Biology
Hai He, Christian Edlich Muth, Steffen N Lindner, Arren Bar-Even
The ribulose monophosphate (RuMP) cycle is a highly efficient route for the assimilation of reduced one-carbon compounds. Despite considerable research, the RuMP cycle has not been fully implemented in model biotechnological organisms such as Escherichia coli, mainly since the heterologous establishment of the pathway requires addressing multiple challenges: sufficient formaldehyde production, efficient formaldehyde assimilation, and sufficient regeneration of the formaldehyde acceptor - ribulose 5-phosphate...
May 14, 2018: ACS Synthetic Biology
Anssi Rantasalo, Joosu Kuivanen, Merja Penttilä, Jussi Jäntti, Dominik Mojzita
Sustainable production of chemicals, materials and pharmaceuticals is increasingly performed by genetically engineered cell factories. Engineering of complex metabolic routes or cell behaviour control systems requires robust and predictable gene expression tools. In this challenging task, orthogonality is a fundamental prerequisite for such tools. In this study, we developed and characterized in depth a comprehensive gene expression toolkit that allows accurate control of gene expression in S. cerevisiae without marked interference with native cellular regulation...
May 11, 2018: ACS Synthetic Biology
Ana Zuniga, Francisco De la Fuente, Fernan Federici, Corinne Lionne, Jerome Bonnet, Victor de Lorenzo, Bernardo Gonzalez
The environmental effects of chemical fertilizers and pesticides have encouraged the quest for new strategies to increase crop productivity with minimal impacts on the natural medium. Plant growth promoting rhizobacteria (PGPR) can contribute to this endeavor by improving fitness through better nutrition acquisition and stress tolerance. Using the neutral (non PGPR) rhizobacterium Cupriavidus pinatubonensis JMP134 as the host, we engineered a regulatory forward loop that triggered the synthesis of the phytohormone indole-3-acetic acid (IAA) in a manner dependent on quorum sensing (QS) signals...
May 10, 2018: ACS Synthetic Biology
Valerie Pezo, Camille Hassan, Dominique Louis, Bruno Sargueil, Piet Herdewijn, Philippe Marliere
We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs...
May 10, 2018: ACS Synthetic Biology
Mathias Foo, Iulia Gherman, Peijun Zhang, Declan G Bates, Katherine Denby
Crop disease leads to significant waste world-wide, both pre- and post-harvest, with subsequent economic and sustainability consequences. Disease outcome is determined both by the plants' response to the pathogen and by the ability of the pathogen to suppress defense responses and manipulate the plant to enhance colonization. The defense response of a plant is characterized by significant transcriptional reprogramming mediated by underlying gene regulatory networks and components of these networks are often targeted by attacking pathogens...
May 10, 2018: ACS Synthetic Biology
Axel Nyström, Antonis Papachristodoulou, Andrew Angel
Introducing synthetic constructs into bacteria often carries a burden that leads to reduced fitness and selective pressure for organisms to mutate their constructs and hence to a reduced functional lifetime. Understanding burden requires suitable methods for accurate measurement and quantification. We develop a dynamic growth model from physiologically relevant first-principles that allows parameters relevant to burden to be extracted from standard growth curves. We test several possibilities for the response of a bacterium to a new environment in terms of resource allocation...
May 10, 2018: ACS Synthetic Biology
Junran Hou, Weiqian Zeng, Yeqing Zong, Zehua Chen, Chensi Miao, Baojun Wang, Chunbo Lou
The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter...
May 8, 2018: ACS Synthetic Biology
Manjunatha Kogenaru, Mark Isalan
Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteins-of-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. The amount of the DD-tagged fusion protein directly impacts its molecular function. Hence, it is important that the background levels be tightly regulated in the absence of any drug. This is especially true for classes of proteins that function at extremely low levels, such as lethality genes involved in tissue development and certain transcriptional activator proteins...
May 7, 2018: ACS Synthetic Biology
Mason R Smith, Stephanie V Tolbert, Fei Wen
Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density...
May 7, 2018: ACS Synthetic Biology
Chelsea Yongxi Hu, Melissa K Takahashi, Yan Zhang, Julius B Lucks
RNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal noise. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs...
May 7, 2018: ACS Synthetic Biology
Marco Santorelli, Daniela Perna, Akihiro Isomura, Immacolata Garzilli, Francesco Annunziata, Lorena Postiglione, Barbara Tumaini, Ryoichiro Kageyama, Diego di Bernardo
The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified 'hybrid' versions of the Hes1 ultradian oscillator combining synthetic and natural parts...
May 4, 2018: ACS Synthetic Biology
Zhongwei Yang, Hongling Wang, Yuxiao Wang, Yuhong Ren, Dong-Zhi Wei
The self-assembly of multi-enzyme into bioreactors is of extensive interest to spatially regulate valuable reactions. Despite the important progresses achieved, methods to precisely manufacture multi-enzymatic complex reactors (MECRs) are still poorly proposed both in vivo and in vitro, particularly for more than three bio-catalytically relevant enzymes. Here, we developed a sequential self-assembly system to form multitude MECRs involving three enzymes in the itaconic acid (IA) pathway with two pairs of protein-peptide interactions...
May 4, 2018: ACS Synthetic Biology
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