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ACS Synthetic Biology

Tomoaki Matsuura, Kazufumi Hosoda, Yoshihiro Shimizu
Robustness against environmental changes is one of the major features of biological systems, but its origin is not well understood. We recently constructed a large-scale computational model of an Escherichia coli-based reconstituted in vitro translation system that enumerates all protein synthesis processes in detail. Our model synthesizes a formyl-Met-Gly-Gly tripeptide (MGG peptide) from 27 initial molecular components through 968 biochemical reactions. Among the 968 kinetic parameters, 483 are non-zero parameters, and the simulator was used to determine how perturbations of 483 individual reactions affect the complex reaction network...
July 13, 2018: ACS Synthetic Biology
Ryan McKay, Pricila Hauk, David Quan, William E Bentley
We generated "sentinel" bacteria that respond to the biomarker nitric oxide (NO) and produce a homogeneous and strong fluorescent response. Our dual-plasmid system consists of a signal "relay" vector that employs an NO-responsive promoter that amplifies the native signal (via expression of T7 Polymerase (T7Pol)) to a second vector responsible for GFP expression. Importantly, to achieve an optimal "sentinel" response, we developed strategies that balance the transcriptional load within cells by altering (i) translation and (ii) activity of the T7Pol...
July 9, 2018: ACS Synthetic Biology
Yusuke Kato
Tight regulatory system for gene expression, which is ideally controlled by unnatural and bio-orthogonal substances, is a keystone for successful construction of synthetic gene circuits. Here, we present a widely applicable approach to construct tight protein translational switches using site-specific unnatural amino acid (Uaa) incorporation systems. As a key mechanism to obtain excellent tightness, we installed gene circuits for positive feedback derepression. This mechanism dramatically suppressed leakage translation in the absence of the Uaa...
July 6, 2018: ACS Synthetic Biology
Louis H Scott, James C Mathews, Gavin R Flematti, Aleksandra Filipovska, Oliver Rackham
Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial genetic circuit allowed the deep mutational scanning of the tetracycline inactivating enzyme TetX, revealing key functional residues. We used both positive and negative selections to confirm the importance of different residues for TetX activity, and profiled activity hotspots for different tetracyclines to reveal substrate-specific activity determinants...
July 6, 2018: ACS Synthetic Biology
Steven Richard Brown, Marta Staff, Rob Lee, John Love, David Parker, Stephen Aves, Thomas Howard
Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyse ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localisation, and enzyme kinetics...
July 6, 2018: ACS Synthetic Biology
Zaigao Tan, James Clomburg, Ramon Gonzalez
Type III polyketide synthases (PKS IIIs) contribute to the synthesis of many economically important natural products, most of which are currently produced by direct extraction from plants or through chemical synthesis. Olivetolic acid (OLA) is a plant secondary metabolite sourced from PKS III catalysis, which along with its prenylated derivatives has various pharmacological activities. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for OLA, here we utilize a synthetic approach to engineer Escherichia coli for the production of OLA...
July 5, 2018: ACS Synthetic Biology
Liting Duan, Jen Marrero Hope, Shunling Guo, Qunxiang Ong, Amaury Francois, Luke Kaplan, Gregory Scherrer, Bianxiao Cui
Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution...
July 5, 2018: ACS Synthetic Biology
Garri Arzumanyan, Kristin N Gabriel, Arjun Ravikumar, Alex A Javanpour, Chang C Liu
The yeast cytoplasmically-localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically increased without influencing genomic replication, thereby supporting in vivo continuous evolution. Here, we report that the pGKL2/TP-DNAP2 plasmid/DNA polymerase pair forms a second orthogonal replication system. We show that custom genes can be encoded and expressed from pGKL2 in a straightforward manner, that error-prone TP-DNAP2s can be engineered, and that pGKL2 replication by TP-DNAP2 is both orthogonal to genomic replication in Saccharomyces cerevisiae and mutually orthogonal with pGKL1 replication by TP-DNAP1...
July 3, 2018: ACS Synthetic Biology
Chang-Hun Ji, Jong-Pyung Kim, Hahk-Soo Kang
Promoter engineering has emerged as a powerful tool to activate transcriptionally silent natural product biosynthetic gene clusters found in bacterial genomes. Since biosynthetic gene clusters are composed of multiple operons, their promoter engineering requires the use of a set of regulatory sequences with a similar level of activities. Although several successful examples of promoter engineering have been reported, its widespread use has been limited due to the lack of a library of regulatory sequences suitable four use in promoter engineering of large, multiple operon-containing biosynthetic gene clusters...
July 2, 2018: ACS Synthetic Biology
Hengji Zhan, Haibiao Xie, Qun Zhou, Yuchen Liu, Weiren Huang
Cancer is still one of the greatest medical challenges in the world. The p53 protein plays an important role in the process of cancer formation. In addition, p53 is found as the most common mutant gene in cancers. Because of the central role of p53 in oncology, it is necessary to construct effective sensors to detect this protein. However, there are few methods to detect the wild type p53 protein or to distinguish the wild type and mutant p53 proteins. In our study, we designed and constructed a p53 genetic sensor that detected the expression of wild type p53 protein in cells...
June 29, 2018: ACS Synthetic Biology
Catarina Coutinho Pacheco, Zsófia Büttel, Filipe Pinto, Guillermo Rodrigo, Javier Carrera, Alfonso Jaramillo, Paula Tamagnini
The use of cell factories for the production of bulk and value-added compounds is nowadays an advantageous alternative to the traditional methods. Nevertheless, the efficiency and productivity of several of these processes can improve with the implementation of microaerobic or anaerobic conditions. In the industrial set, laccases are appealing catalysts able to oxidize a wide range of substrates and reducing O2 to H2 O. In this work, several laccase-based devices were designed and constructed foreseeing the modulation of intracellular oxygen concentration in bacterial chassis...
June 28, 2018: ACS Synthetic Biology
Abayomi Oluwanbe Johnson, Miriam Gonzalez-Villanueva, Kang Lan Tee, Tuck Seng Wong
Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C...
June 27, 2018: ACS Synthetic Biology
Leandro Watanabe, Tramy Nguyen, Michael Zhang, Zach Zundel, Zhen Zhang, Curtis Madsen, Nicholas Roehner, Chris J Myers
The iBioSim tool has been developed to facilitate the design of genetic circuits via a model-based design strategy. This paper illustrates the new features incorporated into the tool for DNA circuit design, design analysis, and design synthesis, all of which can be used in a workflow for the systematic construction of new genetic circuits.
June 26, 2018: ACS Synthetic Biology
Dingyu Liu, Zhitao Mao, Jiaxin Guo, Leyi Wei, Hongwu Ma, Ya-Jie Tang, Tao Chen, Zhiwen Wang, Xueming Zhao
Promoters are among the most important and basic tools for the control of metabolic pathways. However, previous researches mainly focused on screening and characterization of some native promoters in Bacillus subtilis. To develop a broadly applicable promoter system for this important platform organism, we created a de novo synthetic promoter library (SPL) based on consensus sequences by analyzing microarray transcriptome data of Bacillus subtilis 168. A total of 214 potential promoters spanning a gradient of strengths were isolated and characterized by a GFP fluorescence assay...
June 26, 2018: ACS Synthetic Biology
Michael Vilkhovoy, Nicholas Horvath, Che-Hsiao Shih, Joseph A Wayman, Kara Calhoun, James R Swartz, Jeffrey Varner
Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance of E. coli cell-free protein synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function...
June 26, 2018: ACS Synthetic Biology
Franziska Thomas, William Michael Dawson, Eric J M Lang, Antony J Burton, Gail J Bartlett, Guto Rhys, Adrian J Mulholland, Derek N Woolfson
We describe de novo designed α-helical barrels ( HBs) that bind and discriminate between lipophilic biologically active molecules. HBs have five or more α helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric and heptameric HBs bind the environmentally sensitive dye, 1,6-diphenylhexatriene (DPH) in the µM range and fluoresce. Displacement of the dye is used to report the binding of non-fluorescent molecules: palmitic acid and retinol bind to all three HBs with sub-µM inhibitor constants; farnesol binds the hexamer and heptamer; but ß-carotene only binds to the heptamer...
June 26, 2018: ACS Synthetic Biology
Paul D'Agostino, Tobias A M Gulder
The need of new pharmacological lead structures, especially against drug-resistances, has led to a surge in natural product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural products have thus been developed. Direct Pathway Cloning (DiPaC) is an emerging synthetic biology strategy that utilises long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilising T4 DNA polymerase (sequence- and ligation-independent cloning) for gene cluster capture...
June 25, 2018: ACS Synthetic Biology
Christopher Sauer, Emiel Ver Loren van Themaat, Leonie Boender, Daphne Groothuis, Rita Cruz, Leendert Willem Hamoen, Colin Harwood, Tjeerd van Rij
Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12,000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned five orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module...
June 25, 2018: ACS Synthetic Biology
Linyue Zhang, Edward King, Ray Luo, Han Li
Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent D-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH...
June 25, 2018: ACS Synthetic Biology
Ross Kent, Samantha Halliwell, Kate Young, Neil Swainston, Neil Dixon
The ability of RNA to sense, regulate and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5' UTR, by constructing and screening a library of N-terminal synonymous codon variants...
June 21, 2018: ACS Synthetic Biology
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