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ACS Synthetic Biology

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https://www.readbyqxmd.com/read/28602075/a-synthetic-recombinase-based-feedback-loop-results-in-robust-expression
#1
Thomas Folliard, Harrison Steel, Thomas P Prescott, George Wadhams, Lynn J Rothschild, Antonis Papachristodoulou
Accurate control of a biological process is essential for many critical functions in biology, from the cell cycle to proteome regulation. To achieve this, negative feedback is frequently employed to provide a highly robust and reliable output. Feedback is found throughout biology and technology, but due to challenges posed by its implementation, it is yet to be widely adopted in synthetic biology. In this paper we design a synthetic feedback network using a class of recombinase proteins called integrases, which can be re-engineered to flip the orientation of DNA segments in a digital manner...
June 21, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28585817/production-of-medium-chain-fatty-acids-by-yarrowia-lipolytica-combining-molecular-design-and-talen-to-engineer-the-fatty-acid-synthase
#2
Coraline Rigouin, Marc Gueroult, Christian Croux, Gwendoline Dubois, Vinciane Borsenberger, Sophie Barbe, Alain Marty, Fayza Daboussi, Isabelle André, Florence Bordes
Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS...
June 21, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28585808/evidence-for-improved-encapsulated-pathway-behavior-in-a-bacterial-microcompartment-through-shell-protein-engineering
#3
Marilyn F Slininger Lee, Christopher M Jakobson, Danielle Tullman-Ercek
Bacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell...
June 21, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28532147/development-of-a-bacterial-biosensor-for-rapid-screening-of-yeast-p-coumaric-acid-production
#4
Solvej Siedler, Narendar K Khatri, Andrea Zsohár, Inge Kjærbølling, Michael Vogt, Petter Hammar, Christian F Nielsen, Jan Marienhagen, Morten O A Sommer, Haakan N Joensson
Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site...
June 21, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28627886/experimentally-validated-model-enables-debottlenecking-of-in-vitro-protein-synthesis-and-identifies-a-control-shift-under-in-vivo-conditions
#5
Alexander Nieß, Jurek Failmezger, Maike Kuschel, Martin Siemann-Herzberg, Ralf Takors
Cell-free (in vitro) protein systems (CFPS) provide a versatile tool that can be used to investigate different aspects of the transcription-translation machinery by reducing cells to the basic functions of protein formation. Recent improvements in reaction stability and lysate preparation offer the potential to expand the scope of in vitro biosynthesis from a research tool to a multifunctional and versatile platform for protein production and synthetic biology. To date, even the best-performing CFPS are drastically slower than in vivo references...
June 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28602076/rapid-generation-of-universal-synthetic-promoters-for-controlled-gene-expression-in-both-gas-fermenting-and-saccharolytic-clostridium-species
#6
Gaohua Yang, Dechen Jia, Lin Jin, Yuqian Jiang, Yong Wang, Weihong Jiang, Yang Gu
Engineering solventogenic clostridia, a group of important industrial microorganisms, to realize their full potential in biorefinery application is still hindered by the absence of plentiful biological parts. Here, we developed an effective approach for rapid generation of a synthetic promoter library in solventogenic clostridia based on a dual-reporter system (catP-lacZ) and a widely used strong thl promoter. The yielded artificial promoters, spanning 2 orders of magnitude, comprised two modular components (the core promoter region and the spacer between RBS and the translation-initiating code), and the strongest promoter had an over 10-fold-higher activity than the original expression part Pthl...
June 16, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28618222/de-novo-biosynthesis-of-glutarate-via-%C3%AE-keto-acid-carbon-chain-extension-and-decarboxylation-pathway-in-escherichia-coli
#7
Jian Wang, Yifei Wu, Xinxiao Sun, Qipeng Yuan, Yajun Yan
Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the L-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli...
June 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28616968/crispri-srna-transcriptional-translational-regulation-of-extracellular-electron-transfer-in-shewanella-oneidensis
#8
Yingxiu Cao, Xiaofei Li, Feng Li, Hao Song
Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i...
June 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28562030/switching-protein-localization-by-site-directed-rna-editing-under-control-of-light
#9
Paul Vogel, Alfred Hanswillemenke, Thorsten Stafforst
Site directed RNA editing is an engineered tool for the posttranscriptional manipulation of RNA and proteins. Here, we demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization signals, a switch of the subcellular protein localization was achieved. This included the shift from the cytoplasm to the outer-membrane, which typically is inaccessible at the protein-level. Furthermore, the strategy allows to implement photocaging to achieve spatiotemporal control of isoform switching...
June 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28613837/artificial-off-riboswitches-that-downregulate-internal-ribosome-entry-without-hybridization-switches-in-a-eukaryotic-cell-free-translation-system
#10
Atsushi Ogawa, Hiroki Masuoka, Tsubasa Ota
We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide...
June 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28558223/engineered-assimilation-of-exogenous-and-endogenous-formate-in-escherichia-coli
#11
Oren Yishai, Leander Goldbach, Hezi Tenenboim, Steffen N Lindner, Arren Bar-Even
Decoupling biorefineries from land use and agriculture is a major challenge. As formate can be produced from various sources, e.g., electrochemical reduction of CO2, microbial formate-assimilation has the potential to become a sustainable feedstock for the bioindustry. However, organisms that naturally grow on formate are limited by either a low biomass yield or by a narrow product spectrum. The engineering of a model biotechnological microbe for growth on formate via synthetic pathways represents a promising approach to tackle this challenge...
June 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28531348/plant-derived-transcription-factors-for-orthologous-regulation-of-gene-expression-in-the-yeast-saccharomyces-cerevisiae
#12
Gita Naseri, Salma Balazadeh, Fabian Machens, Iman Kamranfar, Katrin Messerschmidt, Bernd Mueller-Roeber
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter...
June 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28594177/a-versatile-toolbox-for-the-control-of-protein-levels-using-n-%C3%AE%C2%B5-acetyl-l-lysine-dependent-amber-suppression
#13
Wolfram Volkwein, Christopher Maier, Ralph Krafczyk, Kirsten Jung, Jürgen Lassak
The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs but approaches based on the exploitation of translational regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of Nε-acetyl-L-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC...
June 8, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28591515/roc-n-ribo-characterizing-a-riboswitching-expression-system-by-modeling-single-cell-data
#14
Christopher Schneider, Leo Bronstein, Jascha Diemer, Heinz Koeppl, Beatrix Suess
RNA-engineered systems offer simple and versatile control over gene expression in many organisms. In particular, the design and implementation of riboswitches presents a unique opportunity to manipulate any reporter device in cis, executing tight temporal and spatial control at low metabolic costs. Assembled to higher order genetic circuits, such riboswitch-regulated devices may efficiently process logical operations. Here, we propose a hierarchical stochastic modeling approach to characterize an in silico repressor gate based on neomycin- and tetracycline-sensitive riboswitches...
June 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28569062/scalable-device-for-automated-microbial-electroporation-in-a-digital-microfluidic-platform
#15
Andrew C Madison, Matthew W Royal, Frederic Vigneault, Liji Chen, Peter B Griffin, Mark Horowitz, George M Church, Richard B Fair
Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet...
June 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28558198/engineered-crispr-systems-for-next-generation-gene-therapies
#16
Michael Pineda, Farzaneh Moghadam, Mo R Ebrahimkhani, Samira Kiani
An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence...
June 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28586214/establishing-an-artificial-pathway-for-de-novo-biosynthesis-of-vanillyl-alcohol-in-escherichia-coli
#17
Zhenya Chen, Xiaolin Shen, Jian Wang, Jia Wang, Ruihua Zhang, Justin Forrest Rey, Qipeng Yuan, Yajun Yan
Vanillyl alcohol is a phenolic alcohol and is used as a flavoring agent in foods and beverages. In this paper, we propose a novel artificial pathway for microbial production of vanillyl alcohol from simple carbon sources. The pathway extends from 4-hydroxybenzoic acid (4-HBA), and needs only three heterologous enzymes, p-hydroxybenzoate hydroxylase (PobA), carboxylic acid reductase (CAR) and caffeate O-methyltransferase (COMT). First, we examined the promiscuous activity of COMT towards 3,4-dihydroxybenzyl alcohol and found a kcat value of 0...
June 6, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28585809/bsim-2-0-an-advanced-agent-based-cell-simulator
#18
Antoni Matyjaszkiewicz, Gianfranco Fiore, Fabio Annunziata, Claire Grierson, Nigel Savery, Lucia Marucci, Mario di Bernardo
Agent-based models (ABMs) provide a number of advantages relative to traditional continuum modelling approaches, permitting incorporation of great detail and realism into simulations, allowing in-silico tracking of single-cell behaviours and correlation of these with emergent effects at the macroscopic level. In this study we present BSim 2.0, a radically new version of BSim, a computational ABM framework for modelling dynamics of bacteria in typical experimental environments including microfluidic chemostats...
June 6, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28571309/multiplex-genome-editing-by-natural-transformation-mugent-for-synthetic-biology-in-vibrio-natriegens
#19
Triana N Dalia, Chelsea A Hayes, Sergey Stolyar, Christopher J Marx, James B McKinlay, Ankur B Dalia
Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented time scales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts...
June 6, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28581725/engineered-control-of-genetic-variability-reveals-interplay-between-quorum-sensing-feedback-regulation-and-biochemical-noise
#20
Yadira Boada, Alejandro Vignoni, Jesus Pico
Stochastic fluctuations in gene expression trigger both beneficial and harmful consequences for cell behavior. Therefore, achieving a desired mean protein expression level while minimizing noise is of interest in many applications, including robust protein production systems in industrial biotechnology. Here we consider a synthetic gene circuit combining intracellular negative feedback and cell-to-cell communication based on quorum sensing. Accounting for both intrinsic and extrinsic noise, stochastic simulations allow us to analyze the capability of the circuit to reduce noise strength as a function of its parameters...
June 5, 2017: ACS Synthetic Biology
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