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Polymerase engineering

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https://www.readbyqxmd.com/read/29142827/overexpression-of-the-primary-sigma-factor-gene-siga-improved-carotenoid-production-by-corynebacterium-glutamicum-application-to-production-of-%C3%AE-carotene-and-the-non-native-linear-c50-carotenoid-bisanhydrobacterioruberin
#1
Hironori Taniguchi, Nadja A Henke, Sabine A E Heider, Volker F Wendisch
Corynebacterium glutamicum shows yellow pigmentation due to biosynthesis of the C50 carotenoid decaprenoxanthin and its glycosides. This bacterium has been engineered for production of various non-native cyclic C40 and C50 carotenoids such as β-carotene, astaxanthin or sarcinaxanthin. In this study, the effect of modulating gene expression more broadly by overexpression of sigma factor genes on carotenoid production by C. glutamicum was characterized. Overexpression of the primary sigma factor gene sigA improved lycopene production by recombinant C...
June 2017: Metabolic Engineering Communications
https://www.readbyqxmd.com/read/29134331/optimization-and-application-of-a-dna-launched-infectious-clone-of-equine-arteritis-virus
#2
Ting Qi, Xiaojun Wang
Reverse genetics is one of the most powerful tools in modern virology. Equine arteritis virus (EAV) is the prototype member of the Equartevirus. In this study, a new reverse genetics system for the recovery of equine arteritis virus from a cDNA plasmid, which contains viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences in both terminals of the viral genome, was developed by optimization of the promoter and terminator regions. Cellular RNA polymerase II drove the transcription of the viral genome...
November 13, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29125739/yield-improvement-of-the-anti-mrsa-antibiotics-wap-8294a-by-crispr-dcas9-combined-with-refactoring-self-protection-genes-in-lysobacter-enzymogenes-oh11
#3
Lingjun Yu, Wei Su, Paul D Fey, Fengquan Liu, Liangcheng Du
The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production...
November 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29121949/quantifying-engineered-nanomaterial-toxicity-comparison-of-common-cytotoxicity-and-gene-expression-measurements
#4
Donald H Atha, Amber Nagy, Andrea Steinbrück, Allison M Dennis, Jennifer A Hollingsworth, Varsha Dua, Rashi Iyer, Bryant C Nelson
BACKGROUND: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks...
November 9, 2017: Journal of Nanobiotechnology
https://www.readbyqxmd.com/read/29119879/magnesium-and-zinc-borate-enhance-osteoblastic-differentiation-of-stem-cells-from-human-exfoliated-deciduous-teeth-in-vitro
#5
Yao-Jen Liu, Wen-Ta Su, Po-Hung Chen
Various biocompatible and biodegradable scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in hard tissue engineering regeneration. We evaluated the distinct effects of magnesium borate, zinc borate, and boric acid blended into chitosan scaffold for osteogenic differentiation of stem cells from exfoliated deciduous teeth. Stem cells from exfoliated deciduous teeth cells are a potential source of functional osteoblasts for applications in bone tissue engineering, but the efficiency of osteoblastic differentiation is low, thereby significantly limiting their clinical applications...
January 1, 2017: Journal of Biomaterials Applications
https://www.readbyqxmd.com/read/29118850/study-of-in-vitro-transcriptional-binding-effects-and-noise-using-constitutive-promoters-combined-with-up-element-sequences-in-escherichia-coli
#6
Qiang Yan, Stephen S Fong
Background: UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results: Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95...
2017: Journal of Biological Engineering
https://www.readbyqxmd.com/read/29115744/engineering-of-a-dna-polymerase-for-direct-m6a-sequencing
#7
Andreas Marx, Joos Aschenbrenner, Stephan Werner, Virginie Marchand, Martina Adam, Yuri Motorin, Mark Helm
Methodologies for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and involved in regulation of gene expression. Current detection techniques are laborious and rely on antibody based enrichment of m6A containing RNA prior to sequencing, as m6A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing...
November 8, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/29100684/high-sensitive-rna-detection-by-one-step-rt-pcr-using-the-genetically-engineered-variant-of-dna-polymerase-with-reverse-transcriptase-activity-from-hyperthermophilies
#8
Hiroyuki Okano, Misato Baba, Katsuhiro Kawato, Ryota Hidese, Itaru Yanagihara, Kenji Kojima, Teisuke Takita, Shinsuke Fujiwara, Kiyoshi Yasukawa
One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4polL329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis...
October 31, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/29098765/enhancing-bone-regeneration-by-combining-mesenchymal-stem-cell-sheets-with-%C3%AE-tcp-col-i-scaffolds
#9
Jun Lin, Jiaqi Shao, Li Juan, Wenke Yu, Xiaojia Song, Pengruofeng Liu, Wenjian Weng, Jinghong Xu, Christian Mehl
The combination of bone marrow-derived mesenchymal stem cells (BMSCs) and biological scaffolds has been demonstrated to be a promising strategy for bone regeneration. However, this method does not result in satisfactory bone regeneration, because the BMSCs are dispersed in the biological scaffolds. The current study developed a new bone regeneration system, which combines synthetic porous three-dimensional scaffolds of β-TCP/COL-I composite with cultured osteogenic sheets of BMSCs. Activity of alkaline phosphatase (ALP), a marker of bone regeneration, was assayed in vitro using enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction...
November 3, 2017: Journal of Biomedical Materials Research. Part B, Applied Biomaterials
https://www.readbyqxmd.com/read/29089766/evaluation-of-synergistic-osteogenesis-between-icariin-and-bmp2-through-a-micro-meso-hierarchical-porous-delivery-system
#10
Qian Wang, Lingyan Cao, Yang Liu, Ao Zheng, Jiannan Wu, Xinquan Jiang, Ping Ji
BMP2 is well known as an outstanding growth factor for inducing new bone formation. However, improvements are still required to use BMP2 effectively and expand its clinical application due to the potential side effects at high doses. In this study, icariin (IC), a type of traditional Chinese medicine, was originally proposed to be a cooperative factor for BMP2. An alkaline phosphatase (ALP) activity assay showed that IC promoted BMP2 osteogenesis in a concentration-dependent manner with significant enhancement at 38...
2017: International Journal of Nanomedicine
https://www.readbyqxmd.com/read/29064377/3d-printing-nano-conductive-multi-walled-carbon-nanotube-scaffolds-for-nerve-regeneration
#11
Se-Jun Lee, Wei Zhu, Margaret Nowicki, Grace Lee, Dong Nyoung Heo, Junghoon Kim, Yi Zuo, Lijie Grace Zhang
In recent years, the development of tissue-engineered 3D scaffolds for various regenerative engineering applications has been widely investigated. Hydrogel polymers are extensively used as tissue engineering scaffold materials due to their unique biocompatible features. However, hydrogel based scaffolds often suffer from weak mechanical strength and a lack of functional groups to enhance cell adhesion. Hence, nanomaterials, such as carbon nanotubes (CNTs), have been introduced to modify the surface properties of scaffolds, thus enhancing the interaction between the neural cells and biomaterials...
October 24, 2017: Journal of Neural Engineering
https://www.readbyqxmd.com/read/29062952/visualized-and-precise-design-of-artificial-small-rnas-for-regulating-t7-rna-polymerase-and-enhancing-recombinant-protein-folding-in-escherichia-coli
#12
Yujia Zhao, Jingjing Fan, Jinlin Li, Jun Li, Xiaohong Zhou, Chun Li
Small non-coding RNAs (sRNAs) have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs) as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs...
December 2016: Synthetic and Systems Biotechnology
https://www.readbyqxmd.com/read/29053685/dna-polymerase-activity-assay-using-near-infrared-fluorescent-labeled-dna-visualized-by-acrylamide-gel-electrophoresis
#13
Eliza L Lewis, Aaron M Leconte
For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis...
October 6, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29051809/impact-of-expansion-and-redifferentiation-under-hypothermia-on-chondrogenic-capacity-of-cultured-human-septal-chondrocytes
#14
Achim von Bomhard, Joseph Faust, Alexander F Elsaesser, Silke Schwarz, Katharina Pippich, Nicole Rotter
A critical limitation in the cultivation of cartilage for tissue engineering is the dedifferentiation in chondrocytes, mainly during in vitro amplification. Despite many previous studies investigating the influence of various conditions, no data exist concerning the effects of hypothermia. Our aim has been to influence chondrocyte dedifferentiation in vitro by hypothermic conditions. Chondrocytes were isolated from cartilage biopsies and seeded in monolayer and in three-dimensional pellet-cultures. Each cell culture was either performed at 32...
January 2017: Journal of Tissue Engineering
https://www.readbyqxmd.com/read/29045151/dynamic-blue-light-inducible-t7-rna-polymerases-opto-t7rnaps-for-precise-spatiotemporal-gene-expression-control
#15
Armin Baumschlager, Stephanie K Aoki, Mustafa Khammash
Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold...
October 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29042614/3d-printed-poly-%C3%AE%C2%B5-caprolactone-scaffold-integrated-with-cell-laden-chitosan-hydrogels-for-bone-tissue-engineering
#16
Liang Dong, Shao-Jie Wang, Xin-Rong Zhao, Yu-Fang Zhu, Jia-Kuo Yu
Synthetic polymeric scaffolds are commonly used in bone tissue engineering (BTE) due to their biocompatibility and adequate mechanical properties. However, their hydrophobicity and the lack of specific cell recognition sites confined their practical application. In this study, to improve the cell seeding efficiency and osteoinductivity, an injectable thermo-sensitive chitosan hydrogel (CSG) was incorporated into a 3D-printed poly(ε-caprolactone) (PCL) scaffold to form a hybrid scaffold. To demonstrate the feasibility of this hybrid system for BTE application, rabbit bone marrow mesenchymal stem cells (BMMSCs) and bone morphogenetic protein-2 (BMP-2) were encapsulated in CSG...
October 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29039590/microrna%C3%A2-218-promotes-prostaglandin-e2-to-inhibit-osteogenic-differentiation-in-synovial-mesenchymal-stem-cells-by-targeting-15%C3%A2-hydroxyprostaglandin-dehydrogenase-nad
#17
Ruijun Cong, Kun Tao, Peiliang Fu, Lieming Lou, Yuchang Zhu, Song Chen, Xinyu Cai, Lingzhou Mao
The chondrogenic differentiation of synovial mesenchymal stem cells (SMSCs) is regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process remain unclear. MicroRNAs (miRs/miRNAs) are undersized non‑coding RNAs responsible for the post‑transcriptional regulation of gene expression, by binding to the 3'‑untranslated regions (3'‑UTRs) of their target mRNAs. miRNAs may constitute a promising tool to regulate SMSC differentiation and to advance the controlled differentiation of SMSCs in therapeutic applications...
December 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/29030273/heterologous-production-of-myxobacterial-%C3%AE-pyrone-antibiotics-in-myxococcus-xanthus
#18
Hilda Sucipto, Domen Pogorevc, Eva Luxenburger, Silke C Wenzel, Rolf Müller
Myxopyronins (MXN) and corallopyronins (COR) are structurally related α-pyrone antibiotics from myxobacteria that represent a highly promising compound class for the development of broad-spectrum antibacterial therapeutic agents. Their ability to inhibit RNA polymerase through interaction with the "switch region", a novel target, distant from previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising candidates for further research. To improve compound supply for further investigation of MXN, COR and novel derivatives of these antibacterial agents, establishment of an efficient and versatile microbial production platform for myxobacterial α-pyrone antibiotics is highly desirable...
October 10, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/29021793/intracellular-crosslinking-of-filoviral-nucleoproteins-with-xintrabodies-restricts-viral-packaging
#19
Tamarand Lee Darling, Laura Jo Sherwood, Andrew Hayhurst
Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs) of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP) along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes. We hypothesized that RNC would be ideal targets for crosslinkers engineered to promote aberrant protein-protein interactions, thereby blocking their orderly transport and packaging...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29020924/plant-jasmonate-zim-domain-genes-shedding-light-on-structure-and-expression-patterns-of-jaz-gene-family-in-sugarcane
#20
Feng Liu, Tingting Sun, Ling Wang, Weihua Su, Shiwu Gao, Yachun Su, Liping Xu, Youxiong Que
BACKGROUND: Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases in the sugarcane industry. Using a molecular biological technique to mine sugarcane resistance genes can provide gene resources for further genetic engineering of sugarcane disease-resistant breeding. Jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins, which involved in the responses to plant pathogens and abiotic stresses, are important signaling molecules of the jasmonic acid (JA) pathway...
October 11, 2017: BMC Genomics
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