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ACS Synthetic Biology

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https://www.readbyqxmd.com/read/28524655/tunable-control-of-an-escherichia-coli-expression-system-for-the-overproduction-of-membrane-proteins-by-titrated-expression-of-a-mutant-lac-repressor
#1
Seong Keun Kim, Dae-Hee Lee, Oh Cheol Kim, Jihyun F Kim, Sung Ho Yoon
Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter...
May 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28520402/catch-and-release-engineered-allosterically-regulated-%C3%AE-roll-peptides-enable-on-off-biomolecular-recognition
#2
Beyza Bulutoglu, Kevin Dooley, Géza R Szilvay, Mark Blenner, Scott Banta
Alternative scaffolds for biomolecular recognition are being developed to overcome some of the limitations associated with immunoglobulin domains. The repeat-in-toxin (RTX) domain is a repeat protein sequence that reversibly adopts the β-roll secondary structure motif specifically upon calcium binding. This conformational change was exploited for controlled biomolecular recognition. Using ribosome display, an RTX peptide library was selected to identify binders to a model protein, lysozyme, exclusively in the folded state of the peptide...
May 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28520394/in-vitro-metabolic-engineering-of-amorpha-4-11-diene-biosynthesis-at-enhanced-rate-and-specific-yield-of-production
#3
Xixian Chen, Congqiang Zhang, Ruiyang Zou, Gregory Stephanopoulos, Heng-Phon Too
In vitro metabolic engineering is an alternative approach to cell-based biosynthesis. It offers unprecedented flexibility for the study of biochemical pathways, thus, providing useful information for the rational design and assembly of reaction modules. Herein, we took the advantage of in vitro metabolic engineering to initially gain insight into the regulatory network of a reconstituted amorpha-4,11-diene (AD) synthetic pathway. Guided by lin-log approximation, we rapidly identified the hitherto unrecognized inhibition of adenosine triphosphate (ATP) on both farnesyl pyrophosphate synthase (FPPS) and amorpha-4,11-diene synthase (ADS)...
May 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28437108/design-of-a-toolbox-of-rna-thermometers
#4
Shaunak Sen, Divyansh Apurva, Rohit Satija, Dan Siegal, Richard M Murray
Biomolecular temperature sensors can be used for efficient control of large-volume bioreactors, for spatiotemporal imaging and control of gene expression, and to engineer robustness to temperature in biomolecular circuit design. Although RNA-based sensors, called "thermometers", have been investigated in both natural and synthetic contexts, an important challenge is to design diverse responses to temperature differing in sensitivity and threshold. We address this issue by constructing a library of RNA thermometers based on thermodynamic computations and experimentally measuring their activities in cell-free biomolecular "breadboards"...
May 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28510446/modularly-constructed-synthetic-granzyme-b-molecule-enables-interrogation-of-intracellular-proteases-for-targeted-cytotoxicity
#5
Patrick Ho, Christopher Ede, Yvonne Y Chen
Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity...
May 16, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28505429/control-of-the-yeast-mating-pathway-by-reconstitution-of-functional-%C3%AE-factor-using-split-intein-catalyzed-reactions
#6
Ka-Hei Siu, Wilfred Chen
Synthetic control strategies using signaling peptides to regulate and coordinate cellular behaviors in multicellular organisms and synthetic consortia remain largely underdeveloped because of the complexities necessitated by heterologous peptide expression. Using recombinant proteins that exploit split intein-mediated reactions, we presented here a new strategy for reconstituting functional signaling peptides capable of eliciting desired cellular responses in S. cerevisiae. These designs can potentially be tailored to any signaling peptides to be reconstituted, as the split inteins are promiscuous and both the peptides and the reactions are amenable to changes by directed evolution and other protein engineering tools, thereby offering a general strategy to implement synthetic control strategies in a large variety of applications...
May 16, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28505410/new-prodigiosin-derivatives-obtained-by-mutasynthesis-in-pseudomonas-putida
#7
Andreas Sebastian Klein, Andreas Domröse, Patrick Bongen, Hannah U C Brass, Thomas Classen, Anita Loeschcke, Thomas Drepper, Luca Laraia, Sonja Sievers, Karl-Erich Jaeger, Jörg Pietruszka
The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid family, which possesses bioactivities as antimicrobial, antitumor and antimalarial agents. Various bacteria including the opportunistic human pathogen Serratia marcescens and different members of the Streptomycetaceae and Pseudoalteromonadaceae produce prodiginines. In addition, these microbes generally accumulate many structurally related alkaloids making efficient prodiginine synthesis and purification difficult and expensive...
May 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28499341/positive-detection-of-gpcr-antagonists-using-a-system-for-inverted-expression-of-a-fluorescent-reporter-gene
#8
Nobuo Fukuda, Misato Kaishima, Jun Ishii, Shinya Honda
The yeast Saccharomyces cerevisiae is a useful eukaryotic host organism for studying GPCRs as monomolecular models. Fluorescent reporter gene assays for GPCRs provide a convenient assay for measuring receptor activity using fluorometric instruments. Generally, these assays detect receptor activation by agonistic ligands as the induction of fluorescent reporter expression, whereas antagonistic activities are detected by competition with agonistic ligands, resulting in decreases in fluorescence intensity. In the current study, we established a system for inverted expression of a fluorescent reporter by incorporating a PEST-tag and finding out a promoter inhibited by activation of the GPCR signaling pathway from yeast endogenous promoters...
May 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28497697/synthetic-protein-scaffolds-for-biosynthetic-pathway-co-localization-on-lipid-droplet-membranes
#9
Jyun-Liang Lin, Jie Zhu, Ian Wheeldon
Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme co-localization and substrate channeling. Here, we develop a protein-based scaffold for co-localizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1)...
May 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28478671/autonomous-cell-migration-to-csf1-sources-via-a-synthetic-protein-based-system
#10
Anam Qudrat, Kevin Truong
Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (i.e., low pH) microenvironment rampant with cytokines, such as CSF1. For potential therapeutic intervention targeted at these CSF1 sources, we have assembled a system of four proteins inside a cell (i.e., HEK293) that initially had no natural CSF1-seeking ability. This system included a newly engineered CSF1 chimera receptor (named CSF1Rchi), the previously engineered Ca(2+) activated RhoA (i...
May 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28463494/sort-seq-approach-to-engineering-a-formaldehyde-inducible-promoter-for-dynamically-regulated-escherichia-coli-growth-on-methanol
#11
Julia Rohlhill, Nicholas R Sandoval, Eleftherios T Papoutsakis
Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence-function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence-function relationships in a robust and rapid way...
May 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28418635/enhancing-protein-production-yield-from-chinese-hamster-ovary-cells-by-crispr-interference
#12
Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, Yu-Chen Hu
Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhfr and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells...
May 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28438021/a-new-improved-and-extended-version-of-the-multicell-bacterial-simulator-gro
#13
Martín Gutiérrez, Paula Gregorio-Godoy, Guillermo Pérez Del Pulgar, Luis E Muñoz, Sandra Sáez, Alfonso Rodríguez-Patón
gro is a cell programming language developed in Klavins Lab for simulating colony growth and cell-cell communication. It is used as a synthetic biology prototyping tool for simulating multicellular biocircuits and microbial consortia. In this work, we present several extensions made to gro that improve the performance of the simulator, make it easier to use, and provide new functionalities. The new version of gro is between 1 and 2 orders of magnitude faster than the original version. It is able to grow microbial colonies with up to 10(5) cells in less than 10 min...
May 8, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28475309/simbiotics-a-multi-scale-integrative-platform-for-3d-modeling-of-bacterial-populations
#14
Jonathan Naylor, Harold Fellermann, Yuchun Ding, Waleed K Mohammed, Nicholas S Jakubovics, Joy Mukherjee, Catherine A Biggs, Phillip C Wright, Natalio Krasnogor
Simbiotics is a spatially explicit multi-scale modeling platform for the design, simulation and analysis of bacterial populations. Systems ranging from planktonic cells and colonies, to biofilm formation and development may be modeled. Representation of biological systems in Simbiotics is flexible, user-defined processes may be in a variety of forms depending on desired model abstraction. Simbiotics provides a library of modules such as cell geometries, physical force dynamics, genetic circuits, metabolic pathways, chemical diffusion and cell interactions...
May 5, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28441472/repurposing-a-two-component-system-based-biosensor-for-the-killing-of-vibrio-cholerae
#15
Premkumar Jayaraman, Maciej B Holowko, Jing Wui Yeoh, Sierin Lim, Chueh Loo Poh
New strategies to control cholera are urgently needed. This study develops an in vitro proof-of-concept sense-and-kill system in a wild-type Escherichia coli strain to target the causative pathogen Vibrio cholerae using a synthetic biology approach. Our engineered E. coli specifically detects V. cholerae via its quorum-sensing molecule CAI-1 and responds by expressing the lysis protein YebF-Art-085, thereby self-lysing to release the killing protein Art-085 to kill V. cholerae. For this report, we individually characterized YebF-Art-085 and Art-085 expression and their activities when coupled to our previously developed V...
May 5, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28459541/decoupling-resource-coupled-gene-expression-in-living-cells
#16
Tatenda Shopera, Lian He, Tolutola Oyetunde, Yinjie J Tang, Tae Seok Moon
Synthetic biology aspires to develop frameworks that enable the construction of complex and reliable gene networks with predictable functionalities. A key limitation is that increasing network complexity increases the demand for cellular resources, potentially causing resource-associated interference among noninteracting circuits. Although recent studies have shown the effects of resource competition on circuit behaviors, mechanisms that decouple such interference remain unclear. Here, we constructed three systems in Escherichia coli, each consisting of two independent circuit modules where the complexity of one module (Circuit 2) was systematically increased while the other (Circuit 1) remained identical...
May 4, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28467058/a-synthetic-alternative-to-canonical-one-carbon-metabolism
#17
Madeleine Bouzon, Alain Perret, Olivier Loreau, Valérie Delmas, Nadia Perchat, Jean Weissenbach, Frédéric Taran, Philippe Marlière
One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct...
May 3, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28437094/rational-design-of-an-ultrasensitive-quorum-sensing-switch
#18
Weiqian Zeng, Pei Du, Qiuli Lou, Lili Wu, Haoqian M Zhang, Chunbo Lou, Hongli Wang, Qi Ouyang
One of the purposes of synthetic biology is to develop rational methods that accelerate the design of genetic circuits, saving time and effort spent on experiments and providing reliably predictable circuit performance. We applied a reverse engineering approach to design an ultrasensitive transcriptional quorum-sensing switch. We want to explore how systems biology can guide synthetic biology in the choice of specific DNA sequences and their regulatory relations to achieve a targeted function. The workflow comprises network enumeration that achieves the target function robustly, experimental restriction of the obtained candidate networks, global parameter optimization via mathematical analysis, selection and engineering of parts based on these calculations, and finally, circuit construction based on the principles of standardization and modularization...
May 2, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28414905/application-of-an-acyl-coa-ligase-from-streptomyces-aizunensis-for-lactam-biosynthesis
#19
Jingwei Zhang, Jesus F Barajas, Mehmet Burdu, George Wang, Edward E Baidoo, Jay D Keasling
ε-Caprolactam and δ-valerolactam are important commodity chemicals used in the manufacture of nylons, with millions of tons produced annually. Biological production of these highly valued chemicals has been limited due to a lack of enzymes that cyclize ω-amino fatty acid precursors to corresponding lactams under ambient conditions. In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize 4-aminobutyric acid into γ-butyrolactam, 5-aminovaleric acid into δ-valerolactam and 6-aminocaproic acid into ε-caprolactam...
April 30, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28426191/genome-wide-abolishment-of-mobile-genetic-elements-using-genome-shuffling-and-crispr-cas-assisted-mage-allows-the-efficient-stabilization-of-a-bacterial-chassis
#20
Kinga Umenhoffer, Gábor Draskovits, Ákos Nyerges, Ildikó Karcagi, Balázs Bogos, Edit Tímár, Bálint Csörgő, Róbert Herczeg, István Nagy, Tamás Fehér, Csaba Pál, György Pósfai
The ideal bacterial chassis provides a simplified, stable and predictable host environment for synthetic biological circuits. Mutability and evolution can, however, compromise stability, leading to deterioration of artificial genetic constructs. By eliminating certain sources of instability, these undesired genetic changes can be mitigated. Specifically, deletion of prophages and insertion sequences, nonessential constituents of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization...
April 26, 2017: ACS Synthetic Biology
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