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ACS Synthetic Biology

Fabio Annunziata, Antoni Matyjaszkiewicz, Gianfranco Fiore, Claire Grierson, Lucia Marucci, Mario di Bernardo, Nigel Savery
In many biotechnological applications, it is useful for gene expression to be regulated by multiple signals, as this allows the programming of complex behaviour. Here we implement, in Escherichia coli, a system that compares the concentration of two signal molecules, and tunes GFP expression proportionally to their relative abundance. The computation is performed via molecular titration between an orthogonal σ factor and its cognate anti-σ factor. We use mathematical modelling and experiments to show that the computation system is predictable and able to adapt GFP expression dynamically to a wide range of combinations of the two signals, and our model qualitatively captures most of these behaviours...
July 19, 2017: ACS Synthetic Biology
Nicole E Nozzi, Anna E Case, Austin L Carroll, Shota Atsumi
Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp...
July 18, 2017: ACS Synthetic Biology
Sean Andrew Higgins, Sorel Ounkap, David F Savage
Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space...
July 14, 2017: ACS Synthetic Biology
Michiko Kimoto, Ichiro Hirao
Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds-Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins...
July 13, 2017: ACS Synthetic Biology
Yi Xiao, Wen Jiang, Fuzhong Zhang
Responding to nitrogen status is essential for all living organisms. Bacteria have evolved various complex and exquisite regulatory systems to control nitrogen metabolism. However, natural nitrogen regulatory systems, owing to their complexity, often function only in their original hosts and do not respond properly when transferred to another species. By harnessing the Lactococcus GlnRA system, we developed a genetically encoded, cross-species ammonium biosensor that displays a dynamic range up to 9-fold upon detection of ammonium ion...
July 13, 2017: ACS Synthetic Biology
Matthew Deaner, Julio Mejia, Hal S Alper
Standard approaches for dCas9-based modification of gene expression are limited in the ability to multiplex targets, establish streamlined cassettes, and utilize commonly studied Pol II promoters. In this work, we re-purpose the dCas9-VPR activator to act as a dual-mode activator/repressor that can be programmed based solely on target position at gene loci. Furthermore, we implement this approach using a streamlined Pol II-ribozyme system that allows expression of many sgRNAs from a single transcript. By "stepping" dCas9-VPR within the promoter region and ORF we create graded activation and repression (respectively) of target genes, allowing precise control over multiplexed gene modulation...
July 12, 2017: ACS Synthetic Biology
Reza M Zadegan, William L Hughes
Self-assembled nucleic acids perform biological, chemical, and mechanical work at the nanoscale. DNA-based molecular machines have been designed here to perform work by reacting with cancer-specific miRNA mimics and then regulating gene expression in vitro by tuning RNA polymerase activity. Because RNA production is topologically restrained, the machines demonstrate chromatin analogous gene expression (CAGE). With modular and tunable design features, CAGE has potential for molecular biology, synthetic biology, and personalized medicine applications...
June 30, 2017: ACS Synthetic Biology
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 30, 2017: ACS Synthetic Biology
Pan Tao, Xiaorong Wu, Wei-Chun Tang, Jingen Zhu, V B Rao
Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes...
June 28, 2017: ACS Synthetic Biology
Fabio Pasin, Leonor C Bedoya, Joan Miquel Bernabé-Orts, Araíz Gallo, Carmen Simón-Mateo, Diego Orzaez, Juan Antonio García
Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids...
June 28, 2017: ACS Synthetic Biology
Sydney E Morris, Aaron W Feldman, Floyd E Romesberg
To bestow cells with novel forms and functions, the goal of synthetic biology, we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts...
June 27, 2017: ACS Synthetic Biology
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 23, 2017: ACS Synthetic Biology
Antoni Matyjaszkiewicz, Gianfranco Fiore, Fabio Annunziata, Claire S Grierson, Nigel J Savery, Lucia Marucci, Mario di Bernardo
Agent-based models (ABMs) provide a number of advantages relative to traditional continuum modeling approaches, permitting incorporation of great detail and realism into simulations, allowing in silico tracking of single-cell behaviors 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 modeling dynamics of bacteria in typical experimental environments including microfluidic chemostats...
June 23, 2017: ACS Synthetic Biology
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
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
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
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
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
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
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
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