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CLONING AND STEM CELLS
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Advanced Macromodel Matrix Structure Cloning for FDTD
PublicationWe propose an improved macromodel-based techniquefor efficient analysis of the structures based on PhotonicCrystals (PhC). The technique involves a new structure of thecoupling matrix and advanced cloning of not only the macromodel matrices, but also the coupling matrices SE and SH.The method allows one to shorten considerably the preprocessingtime, the RAM usage and also the iterating speed of performingFDTD. With this...
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Stem Cells and Cloning-Advances and Applications
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Cloning, Expression and Purification of Wild-Type Trehalose Synthase from Deinococcus geothermalis
PublicationThe aim of this study was isolation and cloning of trehalose synthase gene derived from extremophilic microorganism to the expression vectors in the Tabor-Studier system and its expression in Rosetta(DE3)pLysS Escherichia coli cells. The second phase of the study consisted of proteins purification using an initial denaturation of host proteins and salting-out proteins by ammonium sulfate.
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Cloning, expression, purification and characterization of recombinant trehalose synthase from Deinococcus radiodurans.
PublicationTrehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a nonreducing disacharide in which the two glucose molecules are linked trough a α-1,1-glycosidic bond. Trehalose is readily hydrolyzed to glucose and can be used as a reserve of that sugar in the cell. The presence of trehalose was found in the cells of fungi and yeasts, bacteria, nematodes, insects, eggs, pupae and some plants. The characteristics of trehalose make it...
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Cloning and characterization of the dnaK heat shock operon of the marine bacterium Vibrio harveyi
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Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli.
PublicationGen kodujący alfa-amylazę z Pyrococcus woesei klonowano do plazmidów pET21d(+)lub pYTB2, którymi transformowano komórki Escherichia coli. Otrzymana rekombinantowa alfa amylaza wykazuje maksymalną aktywność przy pH 5.6 w temperaturze 95 stC i przejawia około 24 % początkowej aktywności nawet po 2 godz. inkubacji w 120 stC. Duża termostabilność otrzymanego preparatu świadczy o jego przydatności do enzymatycznego upłynniania skrobi.
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RecA Proteins from Deinococcus geothermalis and Deinococcus murrayi - Cloning, Purification and Biochemical Characterisation
PublicationEscherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS response and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in gamma-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA...
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Recombinant !ermostable AP Exonuclease from Thermoanaerobacter tengcongensis: Cloning, Expression, Purification, Properties and PCR Application
PublicationApurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. !e gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease...
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Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase
PublicationTwo recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ,...
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A new cold-active β-galactosidase from Arthrobacter sp. S3* - gene cloning, overexpression, purification and properties
PublicationA psychrotrophic bacterium producing a cold-active β-galactosidase was isolated from Spitsbergen soil and classified as Arthrobacter sp. S3*. The gene encoding β-galactosidase was isolated from the genomic DNA library, sequenced, cloned, expressed in Escherichia coli, purified by ion exchange chromatography and characterized. The Arthrobaster sp. S3* β-galactosidase is a homotrimeric enzyme composed of 74,4 kDa subunits. It is...
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A novel cold-active beta-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization
PublicationBeta-D-galactosidase (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides. Cold-active beta-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (I) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (II) convert lactose to glucose and galactose which...
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Cloning, expression, and purification of a recombinant cold-adapted β-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b
PublicationAntarktyczny szczep, gram-ujemnych bakterii Pseudoalteromonas sp. 22b, wyizolowanych z przewodu pokarmowego kryla z gatunku Thyssanoessa macrura, jest producentem wewnątrzkomórkowej zimnolubnej beta-galaktozydazy. Białko to jest homotetramerem, w którym każda z jednostek monomeru zbudowana jest z 1028 reszt aminokwasowych. Gen kodujący ten enzym został sklonowany i eksprymowany w komórkach E. coli. Następnie z zastosowaniem procedury...
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Identification, cloning, expression, and characterizationof a highly thermostable single-stranded-DNA-binding protein (SSB) from Deinococcus murrayi
PublicationZidentyfikowano i scharakteryzowano białko SSB-podobne, pochodzące z Deinococcus murrayi (DmuSSB). Otrzymany w reakcji PCR fragment zawierający kompletny gen ssb sklonowano w systemie ekspresyjnym Escherichia coli. Gen składa się z 826 nt, kodujących 276 reszt aminokwasowych z wyliczoną teoretycznie masą cząsteczkową monomeru równą 30,14 kDa. DmuSSB zawiera dwie domeny wiążące jednoniciowe DNA OBna monomer I funkcjonuje jako homodimer....
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Cloning, expression, and biochemical characterization of a coldactive GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil
PublicationAn estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa.Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli...
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THE GENE CLONING, OVEREXPRESSION, PURIFICATION AND BIOCHEMICAL CHARACTERISATION OF A NEW COLD-ADAPTED β–GALACTOSIDASE FROM ARTHROBACTER SP. VII-4
PublicationINTRODUCTION. β-Galactosidase [EC 3.2.1.23] is an enzyme that catalyzes the hydrolysis of O-glycosidic linkages in galactosides. It is commercially used in dairy industry for the production of milk with reduced lactose content. Potentially, the best method for lactose removal under cooling conditions should be carried out with a cold-adapted enzyme. AIM. The aim of this study was to determine the taxonomic affiliation of the isolate...
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A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c - gene cloning, overexpression, purification and properties
PublicationThe development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. In this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological...
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A novel cold-active β-D-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB - gene cloning, purification and characterization
PublicationA gene encoding a novel β-D-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. 32cB was isolated, cloned and expressed in Escherichia coli. The active form of recombinant β-D-galactosidase consists of two subunits with a combined molecular weight of approximately 257 kDa. The enzyme's maximum activity towards o-nitrophenyl-β-D-galactopyranoside was determined as occurring at 28 °C and pH 8.0. However, it...
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A new B-D-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization.
PublicationA psychrotrophic bacterium producing a coldadaptedB-galactosidase upon growth at low temperatureswas classiWed as Arthrobacter sp. 20B. A genomic DNAlibrary of strain 20B introduced into Escherichia coliTOP10F' and screening on X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside)-containing agar plates ledto the isolation of B-galactosidase gene. The B-galactosidasegene (bgaS) encoding a protein of 1,053 amino acids,with a...
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Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with beta-glucosidase, beta-fucosidase and beta-galactosidase activities.
PublicationBackground: Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products...
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Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells
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Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.
Open Research DataEnzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes...
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Comparison of the Ability of Neural Network Model and Humans to Detect a Cloned Voice
PublicationThe vulnerability of the speaker identity verification system to attacks using voice cloning was examined. The research project assumed creating a model for verifying the speaker’s identity based on voice biometrics and then testing its resistance to potential attacks using voice cloning. The Deep Speaker Neural Speaker Embedding System was trained, and the Real-Time Voice Cloning system was employed based on the SV2TTS, Tacotron,...
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Advances in macromodeling technique
PublicationThe paper discuses recent advances in the finite differencetime domain method employing macromodels. New techniquesfor creating irregularly shaped macromodels, grouping ofmacromodels and advanced macromodel cloning are introduced.The last technique is particularly important for efficient analysisof the structures based on Photonic Crystals (PhC). The methodallows one to shorten considerably the preprocessing time, theRAM usage...
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Production of trehalose in a single step enzymatic reaction
PublicationThe aim of this study was isolation and cloning of trehalose synthase gene derived from extremophilic microorganisms Deinoccoci to the expressive E. coli vectors and its biosythesis in different hosts.
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Efficient analysis of structures with rotatable elements using model order reduction
PublicationThis paper presents a novel full-wave technique which allows for a fast 3D finite element analysis of waveguide structures containing rotatable tuning elements of arbitrary shapes. Rotation of these elements changes the resonant frequencies of the structure, which can be used in the tuning process to obtain the S-characteristics desired for the device. For fast commutations of the response as the tuning elements are rotated, the...
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Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae
PublicationGlucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases...
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A New Type of Macro-Elements for Efficient Two-Dimensional FEM Analysis
PublicationThis letter deals with a model order reduction technique applicable for driven and eigenvalue problems solved using the finite element method (FEM). It allows one to efficiently compute electromagnetic parameters of structures comprising small features that require strong local mesh refinement. The subdomains of very fine mesh are separated from the global domain as so called macro-elements that undergo model reduction. The macro-elements...
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Diagonalized Macromodels in Finite Element Method for Fast Electromagnetic Analysis of Waveguide Components
PublicationA new technique of local model-order reduction (MOR) in 3-D finite element method (FEM) for frequency-domain electromagnetic analysis of waveguide components is proposed in this paper. It resolves the problem of increasing solution time of the reduced-order system assembled from macromodels created in the subdomains, into which an analyzed structure is partitioned. This problem becomes particularly relevant for growing size and...
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Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis
PublicationSingle-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism such as DNAreplication, repair, and recombination, and is essential for cell survival. This study reports on the ssb-likegene cloning, gene expression and characterization of a single-stranded DNA-binding protein of Pseudoal-teromonas haloplanktis (PhaSSB) and is the first report of such a protein from psychrophilic microorganism.PhaSSB possesses...
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B-GALACTOSIDASE ARTHROBACTER SP. 32cB - OBTAINING THE GENE SEQUENCE, CONSTRUCTION OF THE EXPRESSION SYSTEM, BIOSYNTHESIS AND BIOCHEMICAL CHARACTERIZATION OF THE ENZYME
PublicationINTRODUCTION: β-Galactosidase is an enzyme which catalyzes the hydrolysis of O glycosidic bond in β-galactosides. Another activity of β galactosidase is a transglycosylation activity. The main industrial use of this protein is the hydrolysis of lactose in milk in a cooling conditions. Synthesis of galactooligosaccharides, which are mostly used as a prebiotics added to some foods or available as dietary supplements, is only one...
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Characterization of a single-stranded DNA-binding-like Protein from Nanoarchaeum equitans - a nucleic acid binding protein with broad substrate specificity
PublicationBackground SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized,...