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ACS Synthetic Biology

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https://www.readbyqxmd.com/read/28106981/controlling-multi-cycle-replication-of-live-attenuated-hiv-1-using-an-unnatural-genetic-switch
#1
Zhe Yuan, Nanxi Wang, Guobin Kang, Wei Niu, Qingsheng Li, Jiantao Guo
A safe and effective human immunodeficiency virus type 1 (HIV-1) vaccine is urgently needed, but remains elusive. While HIV-1 live-attenuated vaccine can provide potent protection as demonstrated in rhesus macaque-simian immunodeficiency virus model, the potential pathogenic consequences associated with the uncontrolled virus replication preclude such vaccine from clinical applications. We investigated a novel approach to address this problem by controlling live-attenuated HIV-1 replication through an unnatural genetic switch that was based on the amber suppression strategy...
January 20, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28103011/discovery-of-a-phosphonoacetic-acid-derived-natural-product-by-pathway-refactoring
#2
Todd S Freestone, Kou-San Ju, Bin Wang, Huimin Zhao
The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28103009/fully-automated-one-step-synthesis-of-single-transcript-talen-pairs-using-a-biological-foundry
#3
Ran Chao, Jing Liang, Ipek Tasan, Tong Si, Linyang Ju, Huimin Zhao
Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present an assembly scheme to synthesize and express a pair of TALENs in a single transcript format with the help of a P2A self-cleavage sequence...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28103008/quantitative-tracking-of-combinatorially-engineered-populations-with-multiplexed-binary-assemblies
#4
Ramsey Zeitoun, Gur Pines, William C Grau, Ryan T Gill
Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory timescales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28102666/a-simulation-approach-for-timing-analysis-of-genetic-logic-circuits
#5
Hasan Baig, Jan Madsen
Constructing genetic logic circuits is an application of synthetic biology, where parts of the DNA of a living cell are engineered to perform a dedicated Boolean function triggered by appropriate concentration levels of certain proteins or by different genetic components. These logic circuits work in a manner similar to electronic logic circuits, but are much more stochastic and hence much harder to characterize. In this paper, we introduce an approach to analyze the threshold value and timing of genetic logic circuits...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28100049/cell-free-translation-is-more-variable-than-transcription
#6
Fabio Chizzolini, Michele Forlin, Noël Yeh Martín, Giuliano Berloffa, Dario Cecchi, Sheref S Mansy
Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, a series of ribosome binding sites (RBS) was investigated. While RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including GC content...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28055179/a-non-natural-protein-rescues-cells-deleted-for-a-key-enzyme-in-central-metabolism
#7
Katherine M Digianantonio, Maria Korolev, Michael H Hecht
An important goal of synthetic biology is to create novel proteins that provide life-sustaining functions in living organisms. Recent attempts to produce novel proteins have focused largely on rational design involving significant computational efforts. In contrast, nature does not design sequences a priori. Instead, nature relies on Darwinian evolution to select biologically functional sequences from nondesigned sequence space. To mimic natural selection in the laboratory, we combed through libraries of novel sequences and selected proteins that rescue E...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28054767/orthogonal-genetic-regulation-in-human-cells-using-chemically-induced-crispr-cas9-activators
#8
Zehua Bao, Surbhi Jain, Valerie Jaroenpuntaruk, Huimin Zhao
The concerted action of multiple genes in a time-dependent manner controls complex cellular phenotypes, yet the temporal regulation of gene expressions is restricted on a single-gene level, which limits our ability to control higher-order gene networks and understand the consequences of multiplex genetic perturbations. Here we developed a system for temporal regulation of multiple genes. This system combines the simplicity of CRISPR/Cas9 activators for orthogonal targeting of multiple genes and the orthogonality of chemically induced dimerizing (CID) proteins for temporal control of CRISPR/Cas9 activator function...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28035820/genetically-encodable-bacterial-flavin-transferase-for-fluorogenic-protein-modification-in-mammalian-cells
#9
Myeong-Gyun Kang, Jumi Park, Gianfranco Balboni, Mi Hee Lim, Changwook Lee, Hyun-Woo Rhee
A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na(+)-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein...
January 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28094993/the-biophysics-of-artificially-expanded-genetic-information-systems-thermodynamics-of-dna-duplexes-containing-matches-and-mismatches-involving-2-amino-3-nitropyridin-6-one-z-and-imidazo-1-2-a-1-3-5-triazin-4-8h-one-p
#10
Xiaoyu Wang, Shuichi Hoshika, Raymond J Peterson, Myong-Jung Kim, Steven A Benner, Jason D Kahn
Synthetic nucleobases presenting non-Watson Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed...
January 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28094982/bacterial-genome-editing-via-a-designed-toxin-antitoxin-cassette
#11
Jie Wu, Aihua Deng, Qinyun Sun, Hua Bai, Zhaopeng Sun, Xiuling Shang, Yun Zhang, Qian Liu, Yong Liang, Shuwen Liu, Yongsheng Che, Tingyi Wen
Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Despite usage of various modules for genomic editing with counter-selectable markers including the toxin genes, an easy-to-use and highly designable toxin-antitoxin (TA) cassette without causing any leakages is urgently needed for efficient genome editing of the Gram-positive bacteria. Here, we reprogramed the bacterial TA systems as a toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, B...
January 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28094975/endoribonuclease-based-two-component-repressor-systems-for-tight-gene-expression-control-in-plants
#12
Yan Liang, Sarah M Richardson, Jingwei Yan, Veronica T Benites, Clarabelle Cheng-Yue, Thu Tran, Jenny Mortimer, Aindrila Mukhopadhyay, Jay D Keasling, Dominique Loque
Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. Meeting these challenges will facilitate transgene expression regulation and support fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression-repression system to tightly control synthesis of transgene products...
January 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28033709/design-and-experimental-validation-of-small-activating-rnas-targeting-an-exogenous-promoter-in-human-cells
#13
Edouard A Harris, Alla Buzina, Jason Moffat, David R McMillen
It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research...
January 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28080041/in-vitro-reconstitution-and-optimization-of-the-entire-pathway-to-convert-glucose-into-fatty-acid
#14
Zheng Liu, Yuchen Zhang, Xiaoge Jia, Mengzhu Hu, Zixin Deng, Yancheng Xu, Tiangang Liu
Glucose and fatty acids play essential physiological roles in nearly all living organisms and the pathway that converts glucose into fatty acid is pivotal to the central metabolic network. We have successfully reconstituted a pathway that converts glucose to fatty acid in vitro using 30 purified proteins. Through systematic titration and optimization of the glycolytic pathway and pyruvate dehydrogenase, we increased the yield of free fatty acid from non-detectable to a level that exceeded 9% of the theoretical yield...
January 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28080037/global-metabolic-engineering-of-glycolytic-pathway-via-multi-copy-integration-in-saccharomyces-cerevisiae
#15
Ryosuke Yamada, Kazuki Wakita, Hiroyasu Ogino
The use of renewable feedstocks for producing biofuels and bio-based chemicals by engineering metabolic pathways of yeast Saccharomyces cerevisiae has recently become an attractive option. Many researchers attempted to increase glucose consumption rate by overexpressing some glycolytic enzymes because most target bio-based chemicals are derived through glycolysis. However these attempts have met with little success. In this study, to create a S. cerevisiae strain with high glucose consumption rate, we used multi-copy integration to develop a global metabolic engineering strategy...
January 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28076956/sbolme-a-repository-of-sbol-parts-for-metabolic-engineering
#16
Hiroyuki Kuwahara, Xuefeng Cui, Ramzan Umarov, Raik Gruenberg, Chris J Myers, Xin Gao
The Synthetic Biology Open Language (SBOL) is a community-driven open language to promote standardization in synthetic biology. To support the use of SBOL in metabolic engineering, we developed SBOLme, the first open-access repository of SBOL 2.0-compliant biochemical parts for a wide range of metabolic engineering applications. The URL of our repository is http://www.cbrc.kaust.edu.sa/sbolme.
January 11, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28052191/engineering-a-thermostable-keto-acid-decarboxylase-using-directed-evolution-and-computationally-directed-protein-design
#17
Lemuel M J Soh, Wai Shun Mak, Paul P Lin, Luo Mi, Frederic Y-H Chen, Robert Damoiseaux, Justin B Siegel, James C Liao
Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened...
January 11, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28073246/a-new-era-of-genome-integration-simply-cut-and-paste
#18
Zihe Liu, Youyun Liang, Ee-Lui Ang, Huimin Zhao
Genome integration is a powerful tool in both basic and applied biological research. However, traditional genome integration, which is typically mediated by homologous recombination, has been constrained by low efficiencies and limited host range. In recent years, the emergence of homing endonucleases and programmable nucleases has greatly enhanced integration efficiencies and allowed alternative integration mechanisms such as non-homologous end joining and microhomology-mediated end joining, enabling integration in hosts deficient in homologous recombination...
January 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28067500/retargeting-a-dual-acting-srna-for-multiple-mrna-transcript-regulation
#19
Ashwin Lahiry, Samuel D Stimple, David W Wood, Richard A Lease
Multi-targeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multi-targeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through simple base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes...
January 9, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28055177/effect-of-genomic-integration-location-on-heterologous-protein-expression-and-metabolic-engineering-in-e-coli
#20
Jacob A Englaender, John Andrew Jones, Brady F Cress, Thomas E Kuhlman, Robert J Linhardt, Mattheos A G Koffas
Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E...
January 5, 2017: ACS Synthetic Biology
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