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ACS Synthetic Biology

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https://www.readbyqxmd.com/read/27797498/reproducibility-of-high-throughput-plate-reader-experiments-in-synthetic-biology
#1
Michael Chavez, Jonathan Ho, Cheemeng Tan
Plate-reader assays are commonly conducted to quantify the performance of synthetic biological systems. However, based on a survey of 100 publications, we find that a majority of the publications does not report critical experimental settings of plate-reader assays, suggesting wide spread issues in their reproducibility. Specifically, critical plate-reader settings, including shaking time and covering method, either vary between labs or are not reported by the publications. Here, we demonstrate that the settings of plate-reader assays have significant impact on bacterial growth, recombinant gene expression, and biofilm formation...
October 31, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27797488/development-of-escherichia-coli-strains-that-withstand-membrane-protein-induced-toxicity-and-achieve-high-level-recombinant-membrane-protein-production
#2
Dimitra Gialama, Kalliopi Kostelidou, Myrsini Michou, Dafni Chrysanthi Delivoria, Fragiskos N Kolisis, Georgios Skretas
Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level MP production...
October 31, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27744683/engineering-modular-biosensors-to-confer-metabolite-responsive-regulation-of-transcription
#3
Andrew K D Younger, Neil C Dalvie, Austin G Rottinghaus, Joshua N Leonard
Efforts to engineer microbial factories have benefitted from mining biological diversity and high throughput synthesis of novel enzymatic pathways, yet screening and optimizing metabolic pathways remain rate-limiting steps. Metabolite-responsive biosensors may help to address these persistent challenges by enabling the monitoring of metabolite levels in individual cells and metabolite-responsive feedback control. We are currently limited to naturally evolved biosensors, which are insufficient for monitoring many metabolites of interest...
October 31, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794611/cyanobacterial-surface-display-system-mediates-engineered-interspecies-and-abiotic-binding
#4
Derek T Fedeson, Daniel C Ducat
Cyanobacteria are uniquely suited for development of sustainable bioproduction platforms, but are currently underutilized in scaled applications in part due to a lack of genetic tools. Here, we develop a surface display system in the cyanobacterial model Synechococcus elongatus PCC7942 via expression of modified versions of the outer membrane porin, SomA. Importantly, we demonstrate accessibility of heterologous functional groups on the recombinant porin to the external environment in living cells. We show that this requires the removal of occluding factors that include lipopolysaccharides and a putative surface layer protein...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794603/engineering-of-taxadiene-synthase-for-improved-selectivity-and-yield-of-a-key-taxol-biosynthetic-intermediate
#5
Steven Edgar, Fu-Shuang Li, Kangjian Qiao, Jing-Ke Weng, Gregory Stephanopoulos
Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodol-ogies, terpene cyclase engineering, P450 engineering, and hydro-lase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Syn-thase, upstream of the promiscuous oxidation step, acts as a practi-cal method for selectivity improvement...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794600/engineered-photoactivatable-genetic-switches-based-on-the-bacterium-phage-t7-rna-polymerase
#6
Tiyun Han, Quan Chen, Haiyan Liu
Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794593/artificial-symmetry-breaking-for-morphogenetic-engineering-bacterial-colonies
#7
Isaac N Nuñez, Tamara F Matute, Ilenne Del Valle, Anton Kan, Atri Choksi, Drew Endy, Jim Haseloff, Timothy Rudge, Fernan Federici
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27794590/synthetic-biogenesis-of-bacterial-amyloid-nanomaterials-with-tunable-inorganic-organic-interfaces-and-electrical-conductivity
#8
Urartu Ozgur Safak Seker, Allen Yuyin Chen, Robert J Citorik, Timothy K Lu
Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires...
October 30, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27783893/the-impact-of-chromatin-dynamics-on-cas9-mediated-genome-editing-in-human-cells
#9
Rene Daer, Josh P Cutts, David A Brafman, Karmella A Haynes
In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear...
October 26, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27775882/application-of-noncanonical-amino-acids-for-protein-labeling-in-a-genomically-recoded-escherichia-coli
#10
Kalle Kipper, Ebba Gregorsson Lundius, Vladimir Curic, Ivana Nikic, Edward A Lemke, Manfred Wiessler, Johan Elf
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells...
October 24, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27744692/recycling-carbon-dioxide-during-xylose-fermentation-by-engineered-saccharomyces-cerevisiae
#11
Peng-Fei Xia, Guo-Chang Zhang, Berkley Walker, Seung-Oh Seo, Suryang Kwak, Jingjing Liu, Heejin Kim, Donald Ort, Shu-Guang Wang, Yong-Su Jin
Global climate change caused by the emission of anthropogenic greenhouse gasses (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling...
October 17, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27744689/dnaplotlib-programmable-visualization-of-genetic-designs-and-associated-data
#12
Bryan S Der, Emerson Glassey, Bryan A Bartley, Casper Enghuus, Daniel B Goodman, D Benjamin Gordon, Christopher A Voigt, Thomas E Gorochowski
DNAplotlib (www.dnaplotlib.org) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both visualization tasks and to extract and overlay associated experimental data. High-quality output is produced in the form of vector-based PDFs, rasterized images, and animated movies. All aspects of the rendering process can be easily customized or extended by the user to cover new forms of genetic part or regulation...
October 17, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27744682/dna-strand-displacement-timer-circuits
#13
Joshua Fern, Dominic Scalise, Angelo Cangialosi, Dylan Howie, Leo Potters, Rebecca Schulman
Chemical circuits can coordinate elaborate sequences of events in cells and tissues, from the self-assembly of biological complexes to the sequence of embryonic development. However, autonomously directing the timing of events in synthetic systems using chemical signals remains challenging. Here we demonstrate that a simple synthetic DNA strand-displacement circuit can release target sequences of DNA into solution at a constant rate after a tunable delay that can range from hours to days. The rates of DNA release can be tuned to the order of 1-100 nM per day...
October 17, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27672702/direct-combinatorial-pathway-optimization
#14
Pieter Coussement, David Bauwens, Jo Maertens, Marjan De Mey
Combinatorial engineering approaches are becoming increasingly popular, yet they are hindered by the lack of specialized techniques for both efficient introduction of sequence variability and assembly of numerous DNA parts, required for the construction of lengthy multigene pathways. In this contribution, we introduce a new combinatorial multigene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly, exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accompanying set of well-characterized and ready-to-use genetic parts, the developed workflow allows effective introduction of various libraries and efficient assembly of multigene pathways...
October 11, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27718551/paired-design-of-dcas9-as-a-systematic-platform-for-the-detection-of-featured-nucleic-acid-sequences-in-pathogenic-strains
#15
Yihao Zhang, Long Qian, Weijia Wei, Yu Wang, Beining Wang, Pingping Lin, Wenchao Liu, Luze Xu, Xiang Li, Dongming Liu, Sida Cheng, Jiaofeng Li, Yixuan Ye, Hang Li, Xiaohan Zhang, Yiming Dong, Xuejin Zhao, Cuihua Liu, Haoqian M Zhang, Qi Ouyang, Chunbo Lou
We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon co-localization of the reporter pair to a ~44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The re-programmability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.
October 10, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27715021/characterization-of-endogenous-and-reduced-promoters-for-oxygen-limited-processes-using-escherichia-coli
#16
Alvaro R Lara, Karim E Jaén, Juan Carlos Sigala, Martina Mühlmann, Lars Regestein, Jochen Büchs
Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker protein, was used as a reporter...
October 7, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27689718/predicting-the-dynamics-and-heterogeneity-of-genomic-dna-content-within-bacterial-populations-across-variable-growth-regimes
#17
Melchior du Lac, Andrew H Scarpelli, Andrew K D Younger, Declan G Bates, Joshua N Leonard
For many applications in microbial synthetic biology, optimizing a desired function requires careful tuning of the degree to which various genes are expressed. One challenge for predicting such effects or interpreting typical characterization experiments is that in bacteria such as E. coli, genome copy number varies widely across different phases and rates of growth, which also impacts how and when genes are expressed from different loci. While such phenomena are relatively well-understood at a mechanistic level, our quantitative understanding of such processes is essentially limited to ideal exponential growth...
October 7, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27648724/expanded-genetic-alphabets-managing-nucleotides-that-lack-tautomeric-protonated-or-deprotonated-versions-complementary-to-natural-nucleotides
#18
Christian B Winiger, Ryan W Shaw, Myong-Jung Kim, Jennifer D Moses, Mariko F Matsuura, Steven A Benner
2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs...
October 7, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27648601/a-general-tool-for-engineering-the-nad-nadp-cofactor-preference-of-oxidoreductases
#19
Jackson K B Cahn, Caroline A Werlang, Armin Baumschlager, Sabine Brinkmann-Chen, Stephen L Mayo, Frances H Arnold
The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity...
October 5, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27700055/controlling-citrate-synthase-expression-by-crispr-cas9-genome-editing-for-n-butanol-production-in-escherichia-coli
#20
Min-Ji Heo, Hwi-Min Jung, Jaeyong Um, Sang-Woo Lee, Min-Kyu Oh
Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under micro-aerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level...
October 4, 2016: ACS Synthetic Biology
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