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ACS Synthetic Biology

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https://www.readbyqxmd.com/read/28812884/controlling-bdellovibrio-bacteriovorus-gene-expression-and-predation-using-synthetic-riboswitches
#1
Mohammed Dwidar, Yohei Yokobayashi
Bdellovibrio bacteriovorus is a predatory bacterium that feeds on Gram-negative bacteria including a wide range of pathogens and thus has potential applications as a biocontrol agent. Owing to its unique life cycle, however, there are limited tools that enable genetic manipulation of B. bacteriovorus. This work describes our first steps toward engineering the predatory bacterium for practical applications by developing basic genetic parts to control gene expression. Specifically, we evaluated four robust promoters that are active during the attack phase of B...
August 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28803472/blue-light-switchable-bacterial-adhesion-as-a-key-step-toward-the-design-of-biofilms
#2
Fei Chen, Seraphine V Wegner
The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light...
August 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28786662/production-of-functional-anti-ebola-antibodies-in-pichia-pastoris
#3
Oliver Purcell, Patrick Opdensteinen, William Chen, Ky Lowenhaupt, Alexander Brown, Mario Hermann, Jicong Cao, Niklas Tenhaef, Eric Kallweit, Robin Kastilan, Anthony J Sinskey, Pablo Perez-Pinera, Johannes F Buyel, Timothy K Lu
The 2013-2016 Ebola outbreak highlighted the limited treatment options and lack of rapid response strategies for emerging pathogen outbreaks. Here, we propose an efficient development cycle using glycoengineered Pichia pastoris to produce monoclonal antibody cocktails against pathogens. To enable rapid genetic engineering of P. pastoris, we introduced a genomic landing pad for reliable recombinase-mediated DNA integration. We then created strains expressing each of the three monoclonal antibodies that comprise the ZMapp cocktail, and demonstrated that the secreted antibodies bind to the Ebola virus glycoprotein by immunofluorescence assay...
August 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28771342/yeast-terminator-function-can-be-modulated-and-designed-on-the-basis-of-predictions-of-nucleosome-occupancy
#4
Nicholas J Morse, Madan R Gopal, James M Wagner, Hal S Alper
The design of improved synthetic parts is a major goal of synthetic biology. Mechanistically, nucleosome occupancy in the 3' terminator region of a gene has been found to correlate with transcriptional expression. Here, we seek to establish a predictive relationship between terminator function and predicted nucleosome positioning to design synthetic terminators in the yeast Saccharomyces cerevisiae. In doing so, terminators improved net protein output from these expression cassettes nearly 4-fold over their original sequence with observed increases in termination efficiency to 96%...
August 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28805373/inefficient-ribosomal-skipping-enables-simultaneous-secretion-and-display-of-proteins-in-saccharomyces-cerevisiae
#5
Carlos A Cruz-Teran, Karthik Tiruthani, Adam Mischler, Balaji M Rao
The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28803482/corynebacterium-glutamicum-chassis-c1-building-and-testing-a-novel-platform-host-for-synthetic-biology-and-industrial-biotechnology
#6
Meike Baumgart, Simon Unthan, Ramona Kloss, Andreas Radek, Tino Polen, Niklas Tenhaef, Moritz Fabian Müller, Andreas Küberl, Daniel Siebert, Natalie Brühl, Kay Marin, Stephan Hans, Reinhard Krämer, Michael Bott, Joern Kalinowski, Wolfgang Wiechert, Gerd Seibold, Julia Frunzke, Christian Rückert, Volker Wendisch, Stephan Noack
Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders and the resulting library of 28 strains was characterized under various environmental conditions...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28803467/not-gate-genetic-circuits-to-control-gene-expression-in-cyanobacteria
#7
Arnaud Taton, Amy T Ma, Mizuho Ota, Susan Golden, James W Golden
To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches. Four NOT gates were tested and characterized in five cyanobacterial strains using fluorescent reporter-gene assays. In comparison to alternative systems used to downregulate gene expression in cyanobacteria, these NOT gates performed well, reducing YFP reporter expression by 4 to 50-fold. We further evaluated these NOT gates by controlling the expression of the ftsZ gene, which encodes a prokaryotic tubulin homolog that is required for cell division and is essential for Synechococcus elongatus PCC 7942...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28803463/bacterial-microcolonies-in-gel-beads-for-high-throughput-screening-of-libraries-in-synthetic-biology
#8
José M Duarte, Içvara Barbier, Yolanda Schaerli
Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g. being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28776984/adoption-of-the-q-transcriptional-system-for-regulating-gene-expression-in-stem-cells
#9
Michael Fitzgerald, Chelsea Gibbs, Adrian A Shimpi, Tara L Deans
The field of mammalian synthetic biology seeks to engineer enabling technologies to create novel approaches for programming cells to probe, perturb, and regulate gene expression with unprecedented precision. To accomplish this, new genetic parts continue to be identified that can be used to build novel genetic circuits to re-engineer cells to perform specific functions. Here, we establish a new transcription-based genetic circuit that combines genes from the quinic acid sensing metabolism of Neorospora crassa and the bacterial Lac repressor system to create a new orthogonal genetic tool to be used in mammalian cells...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28799737/method-to-assemble-genomic-dna-fragments-or-genes-on-human-artificial-chromosome-with-regulated-kinetochore-using-a-multi-integrase-system
#10
Nicholas C O Lee, Jung-Hyun Kim, Nikolai Petrov, Hee-Sheung Lee, Hiroshi Masumoto, William C Earnshaw, Vladimir Larionov, Natalay Kouprina
The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: i) Cloning of large genomic segments; ii) Insertion of multiple DNA blocks at a precise location and iii) The capability to eliminate the assembled region from cells. In this study, we designed the Iterative Integration System (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with Human Artificial Chromosome (HAC) possessing a regulated kinetochore (alphoid(tetO)-HAC)...
August 11, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28796489/rational-modular-rna-engineering-based-on-in-vivo-profiling-of-structural-accessibility
#11
Abigail N Leistra, Paul Amador, Aishwarya Buvanendiran, Alex Moon-Walker, Lydia M Contreras
Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However, development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work, we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system, we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering...
August 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28795570/rapid-and-scalable-preparation-of-bacterial-lysates-for-cell-free-gene-expression
#12
Andriy Didovyk, Taishi Tonooka, Lev Tsimring, Jeff Hasty
Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible...
August 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28749656/development-of-artificial-riboswitches-for-monitoring-of-naringenin-in-vivo
#13
Sungho Jang, Sungyeon Jang, Yu Xiu, Taek Jin Kang, Sang-Hyeup Lee, Mattheos A G Koffas, Gyoo Yeol Jung
Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist...
August 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28793186/an-engineered-optogenetic-switch-for-spatiotemporal-control-of-gene-expression-cell-differentiation-and-tissue-morphogenesis
#14
Lauren Polstein, Mark Juhas, Gabi Hanna, Nenad Bursac, Charles A Gersbach
The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic "on" switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells...
August 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28763198/fundamental-design-principles-for-transcription-factor-based-metabolite-biosensors
#15
Ahmad A Mannan, Di Liu, Fuzhong Zhang, Diego A Oyarzún
Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold...
August 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28783349/orthogonal-ribosome-bio-firewall
#16
Bin Jia, Hao Qi, Bing-Zhi Li, Shuo Pan, Duo Liu, Hong Liu, Yizhi Cai, Ying-Jin Yuan
Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome bio-firewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes...
August 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28771313/a-microfluidic-biodisplay
#17
Francesca Volpetti, Ekaterina Petrova, Sebastian J Maerkl
Synthetically engineered cells are powerful and potentially useful biosensors, but it remains problematic to deploy such systems due to practical difficulties and biosafety concerns. To overcome these hurdles, we developed a microfluidic device that serves as an interface between an engineered cellular system, environment, and user. We created a biodisplay consisting of 768 individually programmable biopixels and demonstrated that it can perform multiplexed, continuous sampling. The biodisplay detected 10 µg/l sodium-arsenite in tap water using a research grade fluorescent microscope, and reported arsenic contamination down to 20 µg/l with an easy to interpret "skull and crossbones" symbol detectable with a low-cost USB microscope or by eye...
August 3, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28771312/multiplexing-engineered-receptors-for-multiparametric-evaluation-of-environmental-ligands
#18
Rachel M Hartfield, Kelly A Schwarz, Joseph J Muldoon, Neda Bagheri, Joshua N Leonard
Engineered cell-based therapies comprise a promising, emerging biomedical technology. Broad utilization of this strategy will require new approaches for implementing sophisticated functional programs, such as sensing and responding to the environment in a defined fashion. Towards this goal, we investigated whether our self-contained receptor and signal transduction system (MESA) could be multiplexed to evaluate extracellular cues, with a focus on elucidating principles governing the integration of such engineered components...
August 3, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28737385/a-synthetic-circuit-for-mercury-bioremediation-using-self-assembling-functional-amyloids
#19
Pei Kun R Tay, Peter Q Nguyen, Neel S Joshi
Synthetic biology approaches to bioremediation are a key sustainable strategy to leverage the self-replicating and programmable aspects of biology for environmental stewardship. The increasing spread of anthropogenic mercury pollution into our habitats and food chains is a pressing concern. Here, we explore the use of programmed bacterial biofilms to aid in the sequestration of mercury. We demonstrate that by integrating a mercury-responsive promoter and an operon encoding a mercury-absorbing self-assembling extracellular protein nanofiber, we can engineer bacteria that can detect and sequester toxic Hg(2+) ions from the environment...
August 2, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28763188/a-tug-of-war-mechanism-for-pattern-formation-in-a-genetic-network
#20
Marcella M Gomez, Murat Arcak
Synthesizing spatial patterns with genetic networks is an ongoing challenge in synthetic biology. A successful demonstration of pattern formation would imply a better understanding of systems in the natural world and advance applications in synthetic biology. In developmental systems, transient patterning may suffice in order to imprint instructions for long-term development. In this paper we show that transient but persistent patterns can emerge from a realizable synthetic gene network based on a toggle switch...
August 1, 2017: ACS Synthetic Biology
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