ArticleMini-prep in ten minutesMarch 1990BioTechniques 8(2):172-3DOI:10.1038/nbt0390-172SourcePubMedAuthors: C ZhouC ZhouThis person is not on ResearchGate, or hasn t claimed this research yet. Y YangY YangThis person is not on ResearchGate, or hasn t claimed this research yet. Ambrose JongChildren s Hospital Los Angeles Request full-text PDFTo read the full-text of this research, you can request a copy directly from the authors.Request full-textDownload citation Copy link Link copied Request full-text Download citation Copy link Link copiedTo read the full-text of this research, you can request a copy directly from the authors.Citations (228) Discover the world s research20+ million members135+ million publications700k+ research projectsJoin for freeNo full-text available To read the full-text of this research, you can request a copy directly from the authors.Request full-text PDFCitations (228)References (0)... Plasmid DNA profile and curing assay for antibiotic resistant bacterial isolates were carried out as described by Birnboin and Doly [16,17,18] and Zhou et al [18] with some modifications. To isolate the plasmids, a volume of nutrient broth was inoculated with aliquot collected from overnight bacterial culture grown in nutrient broth containing antibiotic for 24 h at 37⁰C. ...... Plasmid DNA profile and curing assay for antibiotic resistant bacterial isolates were carried out as described by Birnboin and Doly [16,17,18] and Zhou et al [18] with some modifications. To isolate the plasmids, a volume of nutrient broth was inoculated with aliquot collected from overnight bacterial culture grown in nutrient broth containing antibiotic for 24 h at 37⁰C. ...Cultural, serotyping and plasmid profile of Salmonellae in Lagos, NigeriaArticleFull-text availableApr 2021 Dauphin Dighitoghi MoroProblems associated with typhoid fever epidemic about its diagnosis in developing countries like Nigeria is a perennial healthcare challenge the healthcare sector grapples with. Improper diagnosis of clinical cases have also led to treatment failure and errors as diseases caused by other microorganisms are treated as typhoid fever especially as a result of inadequate reliable diagnostic laboratories. A total of 3,000 stool specimens from patients were analyzed using standard microbiological techniques. Of this, 1,391 Salmonella spp. were recovered, constituting 233 (88.14%) S. typhi while 158 (11.36%) were non-typhoidal Salmonella. S. typhi was recovered from more females, 685(55.6%), than males, 548 (44.4%). The 41 and above age group had the highest incidence of S. typhi of 220(17.8%) in females as against 280 (22.7%) in males within the 21-30 age group. Antibiotic susceptibility testing using the disc diffusion method by Kirby Bauer showed high multiple resistance to most of the 15 different antibiotics tested but susceptible to the first line typhoid fever drugs (chloramphenicol 85%, cotrimoxazole 86.7%, ampicillin 88.3% and amoxicillin 90%) and highly susceptible to third generation cephalosporins and fourth generation fluoroquinolones. The S. typhi tested showed four different resistance patterns. Plasmid profile analysis of 200 multiple antibiotic resistant Salmonella isolates identified culturally and biochemically as S. typhi but by serotyping showed Salmonella other than S. typhi were erroneously classified as S. typhi. Majority of the S. typhi harbored mostly small sized plasmids which ranged from 2.2 Kb to 55.5 Kb. It can be deduced from this study that multiple drug resistance in S. typhi is likely to be plasmid mediated. The eleven antibiotic resistance patterns were reduced to eight plasmid clones indicating the diagnostic efficacy of plasmid profiling over the former method.ViewShow abstract... Plasmid analysis and PCR. Extraction of plasmid DNA was performed by the TENS method (Zhou et al., 1990) with slight modifications. Bacterial cells were grown in Luria Broth (Difco) for 6 h and frozen before starting the protocol, which included at least two steps of treatment with phenol. ...... Plasmid profiles of E. coli 39R861 (lane A) and selected V. vulnificus SerA (lane B, A1-group 1; C, A8-group 2; D, A9-group 3; E, A14-group 4; F, 21A-group 5) and SerE (G, CECT4999) strains. Plasmids were extracted following basically the method ofZhou et al. (1990). ...Electronic Supplementary Material 1DataApr 2008 Guo-Bin HuDan WangChang-Hong WangKun-feng YangPhenotypic and genotypic characterization of a new fish virulent.ViewShow abstract... Two m ethods were utilised to isolate m ini-prep DNA. These m ethods were based on those of Del Sal, 1988, andZhou, 1990, and each produced betw een 5 and 20 pg of plasm id DNA, depending upon the plasm id vector. Both m ethods were used for the rapid isolation of plasm id DNA used for diagnostic restriction endonuclease digestion and in direct plasm id sequencing. ...... TENS m ethod (Zhou, 1990): One and a half mililitres of bacterial overnight culture was spun for 1 0 -2 0 seconds in a bench-top m icrocentrifuge and the culture m edium was poured off leaving 50-100 pi of m edium behind. The cell pellet was resuspended by vortexing. ...Functional and evolutionary analysis of the mouse Muc-1 geneThesisJan 1993Andrew Paul SpicerThe mouse homologue of the human tumour-associated mucin, MUC1, was cloned and full-length sequence was determined. This mucin (previously called polymorphic epithelial mucin) is expressed by the majority of simple secretory epithelial cells in both the mouse and human and is also overexpressed in a large percentage of carcinomas. The mouse gene, Muc-1, encodes an integral membrane protein with 44% of its coding capacity made up of serine, threonine and proline, a composition typical of a highly O-glycosylated protein. The Muc-1 core protein consists of an amino-terminal signal sequence, a repetitive domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing membrane-spanning and cytoplasmic domains. Although overall homology with the human MUC1 protein is only 53%, the transmembrane and cytoplasmic domains exhibit homologies of 90% and 87%, respectively. This level of sequence conservation would suggest that these regions may be functionally important. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat (VNTR) polymorphism. However, this type of polymorphism was found to be present in all other mammalian groups analysed. Data is presented, including sequence obtained for the Muc-1 gene from a large number of species, to suggest how this gene has evolved and to explain possible reasons why the mouse Muc-1 gene does not exhibit minisatellite characteristics. Numerous functions have been suggested for this molecule, yet it still remains unclear what role this protein plays in the tissues and tumours in which it is expressed. In an effort to learn more of the function of the mouse Muc-1 gene, the gene was specifically mutated in embryonic stem (ES) cells. Targeting vectors derived through genomic clones from two strains of mice were utilised and their relative targeting efficiencies are discussed. Several mouse cell lines were created carrying a disruption in the Muc-1 gene. These cell lines were injected into nude mice to create tumours and also injected into blastocysts, in order to generate mice carrying the Muc-1 mutation. These mouse lines will provide a crucial tool in the analysis of the function of this molecule in vivo.ViewShow abstract... The sizes of the different plasmids were estimated based on a control reference strain, P. syringae pv. tomato PT23 that harbors four plasmids (pPT23A, pPT23B, pPT23C and pPT23D that are 100-Kb, 83-Kb, 65-Kb and 36-Kb in size, respectively) [71]. Subsequently, plasmid DNA purification was carried out by equilibrium centrifugation in cesium chlorideethidium bromide gradients (CsCl-EtBr) gradients [72]. ...... For this purpose, genomic DNA was extracted by using the DNeasy tissue kit (QIAGEN Inc., USA) according to the manufacturer s instructions. Furthermore, plasmid DNA mini-preparations were performed using 1.5 mL of an LB broth overnight culture following a modified alkaline lysis method [71]. DNA concentrations and quality from both were determined using NanoDrop ND-1000 (NanoDrop Technologies) spectrophotometer and by agarose gel electrophoresis. ...Complete sequence and comparative genomic analysis of eight native Pseudomonas syringae plasmids belonging to the pPT23A familyArticleFull-text availableMay 2017BMC GENOMICSG.W. Sundin Jose Antonio Gutiérrez Barranquero Francisco M. CazorlaAntonio de VicenteBackgroundThe pPT23A family of plasmids appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. pPT23A-family plasmids (PFPs) are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. The occurrence of repeated sequences including duplicated insertion sequences on PFPs has made obtaining closed plasmid genome sequences difficult. Therefore, our objective was to obtain complete genome sequences from PFPs from divergent P. syringae pathovars and also from strains of P. syringae pv. syringae isolated from different hosts.ResultsThe eight plasmids sequenced ranged in length from 61.6 to 73.8 kb and encoded from 65 to 83 annotated orfs. Virulence genes including type III secretion system effectors were encoded on two plasmids, and one of these, pPt0893-29 from P. syringae pv. tabaci, encoded a wide variety of putative virulence determinants. The PFPs from P. syringae pv. syringae mostly encoded genes of importance to ecological fitness including the rulAB determinant conferring tolerance to ultraviolet radiation. Heavy metal resistance genes encoding resistance to copper and arsenic were also present in a few plasmids. The discovery of part of the chromosomal genomic island GI6 from P. syringae pv. syringae B728a in two PFPs from two P. syringae pv. syringae hosts is further evidence of past intergenetic transfers between plasmid and chromosomal DNA. Phylogenetic analyses also revealed new subgroups of the pPT23A plasmid family and confirmed that plasmid phylogeny is incongruent with P. syringae pathovar or host of isolation. In addition, conserved genes among seven sequenced plasmids within the same phylogenetic group were limited to plasmid-specific functions including maintenance and transfer functions.ConclusionsOur sequence analysis further revealed that PFPs from P. syringae encode suites of accessory genes that are selected at species (universal distribution), pathovar (interpathovar distribution), and population levels (intrapathovar distribution). The conservation of type IV secretion systems encoding conjugation functions also presumably contributes to the distribution of these plasmids within P. syringae populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3763-x) contains supplementary material, which is available to authorized users.ViewShow abstract... TENS mini -prep method was adopted as described by Kado and Liu [17], Ojo and Oso [30], Zhou et al. [46] to extract plasmid DNA of the isolates in the original wastewater sample. ...POTENTIAL EXOELECTROGENIC BACTERIA SPECIES ISOLATED FROM PIGGERY WASTEWATER USED IN GENERATION OF BIOELECTRICITY AND WASTEWATER TREATMENTArticleFull-text availableJan 2017 Toochukwu Ekwutosi OgbulieOnyeka CampbellOgbulie Toochukwu Ekwutosi Henry Uzoma AnuforoThe ability of bacteria in anode chambers of microbial fuel cell (MFC) to transfer electrons from their respiratory chains to anode distinguishes it into mediator or mediator-less MFC. Two groups of 3 MFCs each were constructed with either potassium permanganate as electron acceptor, or potassium ferricyanide. Electrodes used were carbon-carbon (CC), carbon-copper (CCu) and copper-copper (CuCu) in each group. The initial BOD and COD of the piggery wastewater were 420mg/L and 1057mg/L respectively. After 25 days, coulombic efficiency recorded were 69%, 84%, 74%, 76%,72% and 5.10%, while COD removal 65%, 51%, 47%, 83%, 48% and 49% for CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Maximum power density (at Rext = 1000Ω) observed were 79.27mW/m 2 , 156.32mW/m 2 , 92.29mW/m 2 , 60.94mW/m 2 , 39.94mW/m 2 and 14.21mW/m 2 for CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Although Streptococcus sp., Salmonella sp., Lactobacillus sp., Escherichia coli, Proteus mirabilis, Enterobacter sp., Pseudomonas sp., Bacillus sp., Micrococcus luteus, Corynebacterium sp., Shigella sp. and Aeromonas sp. were biochemically identified before treatment of wastewater, but Pseudomonas sp., Escherichia coli, Shigella sp. and Aeromonas sp. did not persist after treatment. Molecular analysis confirmed the absence of Clostridium botulinum, Aeromonas hydrophila, Clostridium butyricum and Rhodobacter ferrireducens, which are known exoelectrogens on the surface of anodes. Plasmid profile revealed that Lactobacillus sp., Proteus mirabilis, Escherichia coli, Pseudomonas sp., Bacillus sp., and Aeromonas sp. carried plasmids. Studies should be undertaken using these persistent bacteria in isolation to ascertain their individual capabilities, together with other cheaper, more environmentally friendly catholytes for better outputs.ViewShow abstract... The presence of plasmid pXap41 was first confirmed in representative strains of X. arboricola pv. pruni with the plasmid profile determined after plasmid DNA extraction, as described in Zhou et al. (1990), and restriction with EcoRI (Fermentas SA, Mont-sur-Lausanne, Switzerland) according to the manufacturer s instructions. ...Complete genome sequence of the stone fruit pathogen Xanthomonas arboricola pv. pruniConference PaperAug 2011 Joël F. Pothier Theo H M Smits Brion Duffy Jochen BlomXanthomonas arboricola pv. pruni (Xap) causes bacterial spot on a wide range of Prunus spp. Outbreaks can result in severe economic losses to fruit quality and yield, branch/tree dieback, and orchard devastation. Xap is endemic in the U.S.A., NZ and is a quarantine regulated pathogen in Europe and elsewhere. Almost nothing is known about the genetics of Xap compared to other xanthomonads. We have sequenced the complete genome of a genotypic-representative Xap strain from Europe (Italy, CFBP 5530). This is the first complete genome sequence for this species. Paired-end 454-pyrosequencing and primer walking on a fosmid library gave 3 contigs. The chromosome is 4.85 Mb with 65.6% GC ratio and 3912 predicted CDS. Xap has a unique 41.2 kb plasmid with a 62.3% GC ratio and 45 CDS. Automatic annotation using several sequenced Xanthomonas genomes as templates, and partial manual annotation, identified a suite of 21 type III secretion system effectors, iron acquisition, and other putative virulence/ecological fitness determinants, many apparently unique to X. arboricola. EDGAR comparisons against X. axonopodis pv. citri str. 306 and X. campestris pv. campestris LMG 8004 indicated a pangenome of 2786 CDS and 848 singletons in Xap.Applied genomics has identified over 90 VNTR, several of which are currently being used for biodiversity analysis of Xap and related subspecies, and design of improved diagnostics.ViewShow abstract... Plasmids were extracted following the method described by [22]. Briefly, cell pellets were obtained from 5 ml of an overnight bacteria broth by centrifugation at 13,000 rpm for 1 min in a centrifuge (Eppendorf 5418, Germany). ...Antibiotics Susceptibility Pattern and Plasmid Profile of Bacteria Isolated from Public Motorcycle HelmetsArticleFull-text availableJul 2016 Obinna T. Ezeokoli Leonard Adamu Faustina Ezeamaramu Jude IyileThe shared use of motorcycle helmets amongst commuters could serve as a source of spread of antibiotic resistant bacteria. In this study, the antibiotic susceptibility pattern and plasmid profile of bacteria isolated from motorcycle helmets in Lagos, Nigeria were investigated. Bacteria were isolated from forty randomly sampled motorcycle helmets and characterized using morphological and biochemical tests. A total of 83 isolates belonging to the phyla Firmicutes (74.7%) and Proteobacteria (25.3%) were obtained, and included species such as Bacillus subtilis, Bacillus anthracis, Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Salmonella spp., Shigellla spp., Staphylococcus aureus, and Staphylococcus spp. We identified species with multiple resistance patterns to commonly used antibiotics such as the β-lactams: augumentin, amoxicillin and cloxacilin, as well as the broad spectrum antibiotics gentamicin. The calculated multiple antibiotic resistance index ranged from 0.3 to 1.0. A number of the isolated species had plasmid DNA which on curing, influenced the overall sensitivity of bacteria to antibiotics. These results suggests (without outright proof) presence of antibiotic resistant plasmids in commercial motorcycle helmets and points to the possible role of plasmids in the response of bacteria to antibiotics tested. Findings of this study further highlights the epidemiological significance of motorcycle helmets sharing amongst commuters.ViewShow abstract... The alkaline lysis method was used to extract bacterial DNA from all bioaerosol samples (Zhou et al., 1990) for Illumina sequencing. Briefly, the cells were pelleted and then lysed in fresh TENS lysis buffer containing 0.1 M NaOH, 1x TE buffer and 0.5% SDS. ...Characterization of the fungal microbiome aerosolized in a residential shower unitArticleJan 2012 Sungwoo Bae Kerry A Kinney Maria D King Juan P. MaestreAlthough human exposure to water aerosols is common in residential showers, the droplet distributionpatterns generated in showers are not well understood nor is the bacteria released during showeroperation. In this study, a two-phase flow Particle Tracking Velocimetry (PTV) algorithm was successfullyused to characterize the spatial spray pattern and velocity field in two experimental showers (one lowflowand one high-flow). In addition, the airborne bacteria present in the shower over nearly 5 months ofcontrolled operation was determined for both showers. The results indicate that the droplet velocity outof the low-flow showerhead (which had fewer orifices) was significantly higher than that out of the highflowshowerhead resulting in a higher aerosol number concentration in the low-flow shower and moreconsistent wetting of the shower wall. Both showerheads generated droplets in the respirable range andgenera of potential health concern were observed in the shower aerosols measured both prior to andfollowing shower operation. The study provides one of the first visualizations of droplet spray patterns inresidential showers and provides insight into the airborne bacteria present in showers.ViewShow abstract... Plates were incubated at 378C for 24 h. Ampicillinresistant colonies were picked, grown overnight in~2 mL liquid LB, and their plasmid DNA was isolated by small-scale DNA preparation (Zhou et al., 1990). Plasmid DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. ...N- and C-terminal non-conserved residues contribute to transactivation by a sea anemone ( Nematostella vectensis ) NF-κB transcription factorArticleFull-text availableDec 2015BIOS Anar AlshanbayevaAjith ThomasMartine TremblayThomas D. GilmoreNF-κB is an evolutionarily conserved eukaryotic transcription factor that plays a role in many important developmental and immune-related processes by activating target gene expression. The goal of these experiments was to define the sequences required for a sea anemone NF-κB s intrinsic transactivation activity by using mutant proteins with serial deletions of the N- and C-terminal sequences. Deletion mutants were constructed that were missing the C-terminal 15, 32 or 47 amino acids (aa) or the N-terminal 17, 27 or 47 aa of the 440 aa NF-κB protein from the starlet sea anemone, Nematostella vectensis (Nv), a simple model organism in the phylum Cnidaria. These Nv-NF-κB mutants were expressed as GAL4 fusion proteins in yeast, and their transactivation activities were assessed by LacZ reporter gene assays. The deletion of 47 aa from either the N terminus or the C terminus of NF-κB completely inactivates the transactivation function of Nv-NF-κB. In addition, we identified proline-258 in the center of the protein as a key residue for the transactivation function of Nv-NF-κB. Taken together, these results demonstrate that non-conserved N- and C-terminal residues are both required for the transcriptional activating function of the sea anemone NF-κB protein, suggesting that it has a novel functional domain structure among known NF-κB proteins.ViewShow abstract... The alkaline lysis method was used to extract bacterial DNA from all bioaerosol samples (Zhou et al., 1990) for Illumina sequencing. Briefly, the cells were pelleted and then lysed in fresh TENS lysis buffer containing 0.1 M NaOH, 1x TE buffer and 0.5% SDS. ...Characterization of the microbial community aerosolized in showersArticleJan 2014A. Caya Chloe Wooldridge Maria D King Kerry A KinneyView... HCl (pH 7.5), 70 mM-MgCl,, 1 M-KCl, 20 mM-2-mercaptoethanol) was generally used for all restrictions. Miniprep DNA for restriction analysis and cloning or sequencing purposes was isolated by a modification of the method of Zhou et al. (1990). This initially involved harvesting 1.5 ml of an overnight culture by centrifuging in a microfuge for 30 s. ...Molecular Cloning and Analysis of the Gene Encoding the NADH Oxidase from Streptococcus faecalis 10C1. Comparison with NADH Peroxidase and the Flavoprotein Disulfide ReductasesArticleFull-text availableJan 1992J MOL BIOLR. Paul Ross Al ClaiborneThe gene encoding the streptococcal flavoprotein NADH oxidase (NOXase), which catalyzes the four-electron reduction of 0,-+ 2H,O, has been cloned and sequenced from the genome of Streptococcus (Enterococcus) fuecaEis lOC1 (ATCC 11700). The deduced NOXase protein sequence corresponds to a molecular mass of 48.9 kDa and contains three previously sequenced cysteinyl peptides obtained with the purified enzyme. In Escherichia coli, the expressed nox gene produced a catalytically active product, which retained it,s immunoreactivity to affinity-purified NOXase antisera. Alignment of the NOXase protein sequence with that of streptococcal NADH peroxidase (NPXase) revealed that the proteins are 44% identical. Among the most highly conserved segments is a sequence containing Cys42; this residue is known to exist as a stabilized cysteine-sulfenic acid (Cys-SOH) in NPXase and serves as the non-flavin redox center. In addition, three previously identified NPXase segments, known to be involved in FAD and NAD(P)-binding in other pyridine nucleotide-linked flavoprotein oxidoreductases, are strongly conserved in NOXase. Overall. the extensive homology observed between NOXase and NPXase suggests that the monomer chain fold of the oxidase closely resembles that of the peroxidase. Roth sequences share limited but significant homology to those of glutathione reductase and other members of bhr flavoprotein disulfide reductase family. These and other considerations suggest that these two unusual streptococcal flavoproteins constitute a distinct class of FAD-dependent oxidoreductases, the flavoprotein peroxide reductases, easily contrasted with enzymes such as glutathione reductase and thioredoxin reductase.ViewShow abstract... Maize, plasmid, and phage DNAs were isolated by the cetyltrimethylammonium bromide (Rogers and Bendich, 1985), alkaline lysis (Zhou, 1990), and plate lysis (Maniatis et al., 1982) methods, respectively. ...The glossy1 Locus of Maize and an Epidermis-Specific cDNA from Kleinia odora Define a Class of Receptor-Like Proteins Required for the Normal Accumulation of Cuticular WaxesArticleApr 1997PLANT PHYSIOLJ. D. HansenMutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).ViewShow abstract... The efficiency of any subsequent enzyme-catalyzed reaction is predicated upon the concentrations of DNA substrates and inhibitors, including undesired DNA (16,17). Most molecular biologists now rely upon DNA purification kits based on silica, or less frequently ion exchange resins, as opposed to the phenol/chloroform extractions (18), alcohol precipitations (19), and cesium chloride gradient ultra centrifugations that were common in the past (20). Anion exchange chromatography is time-consuming, so it is generally employed only in those instances where the purity of plasmids isolated from cell extracts is essential, such as mammalian cell transfection. ...Why Johnny can t clone: Common pitfalls and not so common solutionsArticleFull-text availableSep 2015 Ichiro MatsumuraThe demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of traditional ligation-dependent cloning are non-trivial, most can be avoided with sufficient effort and attention to detail. Here, the chemical properties of enzymes and reagents used to clone genes into plasmids are reviewed to draw attention to the most pertinent details. In particular, the virtues of agarose gel electrophoresis monitoring, the nature of the interactions between DNA and silica, and challenges associated with thermostable DNA polymerases, restriction endonucleases, and T4 DNA ligase are explored. Common pitfalls associated with Escherichia coli transformation and DNA modifying enzymes are also described. A thorough understanding of established methods is essential for troubleshooting, implementing alternative approaches, and inventing new techniques in response to changes in technology and demand.ViewShow abstract... Miniprep DNA samples were prepared using a modified alkaline lysis procedure (Zhou et al., 1990). Other recombinant DNA techniques were performed essentially as described previously (Sambrook et al., 1989). ...Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesisArticleDec 1994J CELL BIOLJohn A. HeymanSeveral groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or peroxisome ghosts. These ghosts proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.ViewShow abstract... E. amylovora strains were grown on nutrient agar with 5 % (w/v) sucrose at 26 °C. For plasmid isolation, 162 strains originating from Poland were grown in CIRCLEGROW ® (MP Biochemicals) liquid medium and crude plasmid DNA was isolated with the method of Zhou et al. (1990). Isolated plasmid DNA was digested with BamHI, and restriction profiles were examined with gel electrophoresis (1 % agarose). ...A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmidsArticleFull-text availableDec 2014Arch Microbiol Emadeldeen Ismail Jochen Blom Alain Bultreys Joanna PuławskaRecent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.ViewShow abstract... shaking at 250 rpm. The plasmid DNAs were extracted from 1.5 ml of the overnight cultures using a rapid phenol-free extraction procedure (Zhou, 1990) and screened for the correct DNA inserts by restriction analyses with various endonucleases. Remaining overnight cultures (1.5 ml) of correct recombinant plasmids were purified using the Aurum™ Plasmid Mini Kit (Bio-Rad Laboratories, Hercules, CA) and the DNA inserts were sequenced. ...CHARACTERIZATION OF TWO NOVEL MARINE CALICIVIRUSES: MOLECULAR AND SEROLOGICAL APPROACHES FOR IMPROVED DIAGNOSTICSArticleShasta D McClenahanView... Colonies were grown overnight as minicultures, in 3 ml of 2XYT medium containing ampicillin (100 µg/ml), while shaken at 270 rpm at 37°C. Plasmid DNA was extracted from 1.5 ml of the minicultures using a phenol-free method (Zhou et al., 1990). To screen for recombinant plasmids, plasmid DNAs were digested with restriction enzymes HindIII, EcoRI, ApaI, BamHI, and a combination of ...DETECTION AND MOLECULAR CHARACTERIZATION OF CETACEAN AND PINNIPED POXVIRUSES ASSOCIATED WITH CUTANEOUS LESIONSArticleFull-text available Alexa J BrachtView... Two methods were used for small-scale isolation of plasmids, namely the TENS and the Mini-prep methods, respectively (Sambrook et al., 1989;Zhou et al., 1990). ...The Construction and Phenotypic Characterization of Mycobacterial Mutants Deficient in DNA GlycosylasesArticleFull-text available Vivianne J GoosensView... Plasmid DNAwas isolated according to a modified alkaline lysis method [7,38] and separated by electrophoresis on 0.6% agarose gels [24]. Plasmid size was estimated by comparison with plasmids from Pseudomonas syringae pv. ...62-kb Plasmids Harboring rulAB Homologues ConferDataFull-text availableAug 2014 Francisco M. CazorlaJ. C. CodinaC AbadAntonio de VicenteView... The alkaline lysis method was used to extract bacterial DNA from all bioaerosol samples (Zhou et al., 1990) for Illumina sequencing. Briefly, the cells were pelleted and then lysed in fresh TENS lysis buffer containing 0.1 M NaOH, 1x TE buffer and 0.5% SDS. ...Impact of Sampler Selection on the Characterization of the Indoor Microbiome via High-Throughput Sequencing,” by Andrew Hoisington, Juan P. Maestre, Maria King, Jeffrey Siegel, and Kerry Kinney.ArticleApr 2014BUILD ENVIRON Andrew Hoisington Juan P. Maestre Maria D King Kerry A KinneyView... As a result, cloning with the pGEM-T Vector System I (Promega) was required to isolate each sequence; we followed the protocol provided by the manufacturer. Miniprep followed Zhou et al. [31], and the sequencing reactions were prepared as described before. Samples that have succeeded in sequencing were deposited in GenBank (accession codes KX212263-KX212309, Ilhéus, Rio Claro, São João da Boa Vista and Santa Rita do Passa Quatro colonies). ...Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae)ArticleFull-text availableMay 2017Curr Microbiol Manuela Oliveira RamalhoCintia MartinsLarissa Marin Rodrigues da Silva Odair BuenoThis study focuses on the weaver ant, Camponotus textor, Forel which occurs in some areas of the Brazilian Cerrado and Atlantic Forest, and its symbionts: Blochmannia, an obligate symbiont of Camponotus, and Wolbachia, known for causing reproductive alterations in their hosts. The main goal of this study was to investigate the presence, frequency of occurrence, and diversity of Wolbachia and Blochmannia strains in C. textor colonies. We found high infection rates (100%) and the occurrence of at least two distinct strains of Blochmannia (H_1 or H_7) in the same species. The observed haplotype variation within a single species may result from the high mutation rate of the symbiont. Similarly, the Wolbachia was found in all colonies with different rates of infections and a new strain (supergroup A) was deposited in the MLST database. The diversity found in the present study shows that there is still much to explore to understand about these symbiotic interactions.ViewShow abstract... Copper resistance and plasmid profile. Plasmid DNA from the selected strains (Table 1) was isolated according to a modified alkaline lysis method (17,78) and separated by electrophoresis on 0.6% agarose gels. Plasmid size was estimated by comparison with the control strain P. syringae pv. ...A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin ProductionArticleFull-text availableNov 2013PHYTOPATHOLOGY Jose Antonio Gutiérrez Barranquero Victor Carrion Jesús MurilloAntonio de VicenteABSTRACT Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.ViewShow abstract... Los posibles ATGs se identificaron traduciendo los ESTs con ExPASy. Para verificar el tamaño de los cDNAs clonados, los plásmidos extraídos según el método de lisis alcalina (Zhou et al., 1990) se digirieron con la enzima BsrGI (Cat. No. R0575S, BioLabs) para escindir los cDNAs del vector pDONR222 donde se encuentran clonados (Martínez-Hernández et al., 2010). ...IDENTIFICATION OF cDNAs FROM Agave tequilana Weber Var. azul WITH SIMILARITY TO FUROSTANOL GLYCOSIDE 26-O-Β-GLUCOSIDASESArticleFull-text availableDec 2019REV FITOTEC MEX Aida Martinez-Hernandez Zurisadai MonroySUMMARY Furostanol glycoside 26-O-β-glucosidase (F26G) participates in the last step of steroidal saponin biosynthesis, cleaving the glucose bound to the C26 of the hydroxylin furostanol glucoside to allow the formation of spirostane. Despite the existence of numerous studies showing the structural diversity and biological activity of plant saponins, as well as their recognized biotechnological and pharmacological potential, few enzymes involved in the biosynthetic pathway of steroidal saponins have been studied and only one native F26G enzyme has been isolated and functionally characterized. In this study, by searching a database of Agave tequilana Weber var. azul (Agave DB), 46 ESTs (Expressed Sequence Tags) of complementary DNA (cDNAs) similar to the known F26G were identified through BLASTX, most come from cDNA from floral tissues (anthers and ovaries) or roots; both organs are saponins producers. Among them, six ESTs were identified whose alignment indicates that they represent three cDNAs different from each other. These ESTs were submitted to the dbEST database of NCBI (National Center for Biotechnology Information), being this the first record of native cDNA sequences putatively encoding F26G-like in Agave. The size of the cloned cDNAs and their alignment to the 5 end of the known F26G suggest they are full-length cDNAs. Specific differential primers were designed for each type of cDNA and their expression in vegetative tissues of A. tequilana was analyzed by RT-PCR. Quantitative PCR protocols (qPCR) were established for future studies on gene expression regulation. The information reported here will serve as a basis for studying the function, regulation and enzymatic activity of F26G-like cloned from Agave.RESUMEN La furostanol glicósido 26-O-β-glucosidasa (F26G) participa en el último paso de la biosíntesis de saponinas esteroidales, lo que escinde la glucosa unida al hidroxilo del C26 del furostanol glucósido para permitir la formación del espirostano. A pesar de la existencia de numerosos estudios que muestran la diversidad estructural y actividad biológica de las saponinas de plantas, así como su reconocido potencial biotecnológico y farmacológico, pocas enzimas participantes en la ruta de biosíntesis de las saponinas esteroidales han sido estudiadas y sólo una enzima nativa tipo F26G ha sido purificada y caracterizada funcionalmente. En este estudio, mediante búsqueda por BLASTX en una base de datos de Agave tequilana Weber var. azul (Agave DB), se identificaron 46 ESTs (Expressed Sequence Tags) de DNA complementario (cDNAs) similares a la F26G de Asparagus officinalis, la mayoría provienen de cDNAs clonados a partir de tejidos florales (anteras y ovarios) o raíz; ambos órganos son productores de saponinas. Entre ellos, se identificaron seis ESTs cuyo alineamiento indica que representan a tres cDNAs diferentes entre sí. Estos ESTs se registraron en la base de datos dbEST de NCBI (National Center for Biotechnology Information), el cual fue el primer registro de secuencias nativas de cDNAs potencialmente codificantes similares a F26G en Agave. El tamaño de los cDNAs clonados y su alineamiento hacia el lado 5 de las F26G conocidas sugieren que son cDNAs completos. Se diseñaron iniciadores diferenciales específicos para cada tipo de cDNA y se analizó por RT-PCR su expresión en tejidos vegetativos de A. tequilana. Se establecieron protocolos de PCR cuantitativo (qPCR) para estudios posteriores de regulación de su expresión génica. La información aquí reportada servirá como base para estudiar la función, regulación y actividad enzimática de las enzimas clonadas de Agave similares a F26G. Palabras clave: Agave tequilana, cDNA, EST, furostanol glicósido 26-O-β-glucosidasa, saponinas.ViewShow abstract... TENS mini -prep method was adopted as described by Kado and Liu [17], Ojo and Oso [30], Zhou et al. [46] to extract plasmid DNA of the isolates in the original wastewater sample. ...Potential exoelectrogenic bacteria species isolated from piggery wastewater used in generation of bioelectricity and wastewater treatmentArticleFull-text availableJan 2017 Ogbulie Te Henry Uzoma Anuforo Campbell AkujobiW.O. OkikaThe ability of bacteria in anode chambers of microbial fuel cell (MFC) to transfer electrons from their respiratory chains to anode distinguishes it into mediator or mediator-less MFC. Two groups of 3 MFCs each were constructed with either potassium permanganate as electron acceptor, or potassium ferricyanide. Electrodes used were carbon - carbon (CC), carbon -copper (CCu) and copper - copper (CuCu) in each group. The initial BOD and COD of the piggery wastewater were 420mg/L and 1057mg/L respectively. After 25 days, coulombic efficiency recorded were 69%, 84%, 74%, 76%,72% and 5.10%, while COD removal 65%, 51%, 47%, 83%, 48% and 49% for CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Maximum power density (at Rext = 1000ω) observed were 79.27mW/m², 156.32mW/m², 92.29mW/m², 60.94mW/m², 39.94mW/m² and 14.21mW/m²for CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Although Streptococcus sp., Salmonella sp., Lactobacillus sp., Escherichia coli, Proteus mirabilis, Enterobacter sp., Pseudomonas sp., Bacillus sp., Micrococcus luteus, Corynebacterium sp., Shigella sp. And Aeromonas sp. Were biochemically identified before treatment of wastewater, but Pseudomonas sp., Escherichia coli, Shigella sp. And Aeromonas sp. Did not persist after treatment. Molecular analysis confirmed the absence of Clostridium botulinum, Aeromonas hydrophila, Clostridium butyricum and Rhodobacter ferrireducens, which are known exoelectrogens on the surface of anodes. Plasmid profile revealed that Lactobacillus sp., Proteus mirabilis, Escherichia coli, Pseudomonas sp., Bacillus sp., and Aeromonas sp. Carried plasmids. Studies should be undertaken using these persistent bacteria in isolation to ascertain their individual capabilities, together with other cheaper, more environmentally friendly catholytes for better outputs.ViewShow abstract... The procedure uses the principle of enzyme cleavage and cell lysis. This method does not include the use of lysozyme nor phenolchloroform (Zhou et al., 1990;Shahriar et al., 2011). ...Screening of selected grains and cashew nuts in South East Nigeria for aflatoxigenic fungi by multiplex PCR techniqueArticleFull-text availableApr 2020Nwachukwu Ikenna Christian Chibuzor OpurumEkwe David OnuContamination of feeds by aflatoxins has been of global concern as a result of their noxious effects on human and animal health as well as their importance in international trade. Timely assessment of fungal contaminants, characterization and identification of the mycotoxigenic species are very necessary, not just for assessing food quality, but also for the development of strategies to control and ensure food safety. In this study, fungal species isolated from selected grains and cashew nut samples from south east Nigeria were screened for aflatoxigenic potential by Multiplex PCR Technique. Primers were designed specifically for the amplification of genes responsible for aflatoxigenic biosynthesis pathways. The necessary experimental conditions were standardized to achieve optimum mPCR result. The DNA extracted from seven fungal isolates were subjected to multiplex PCR using nor-1 (F R), ver-1 (F R), omtA (F R) and aflR (F R) as the primer pairs. DNA samples from AF4 and AP1 possessed the four genes needed for aflatoxigenic biosynthesis pathways. AF1 and AP3 did not show any positive reaction to the four genes after amplification following PCR. However, AF2 and AF3 showed positive response to the two structural genes (nor-1and ver-1) and also for the regulatory gene (aflR) but were negative to the omtA structural gene. The DNA sample of AP3 was only reactive to the structural gene aflR but, did not have the 3 structural genes. Multiple PCR assay has proven to a more reliable, accurate and sensitive molecular tool for the detection and characterization of mycotoxin-producing fungi from agricultural commodities.ViewShow abstract... The alkaline lysis method was used to extract bacterial DNA from all bioaerosol samples (Zhou et al., 1990) for Illumina sequencing. Briefly, the cells were pelleted and then lysed in fresh TENS lysis buffer containing 0.1 M NaOH, 1x TE buffer and 0.5% SDS. ...Droplet distribution and airborne bacteria in an experimental shower unitArticleNov 2017WATER RESC.E. Estrada-Perez Maria D King Kerry A Kinney Juan P. MaestreAlthough human exposure to water aerosols is common in residential showers, the droplet distribution patterns generated in showers are not well understood nor is the bacteria released during shower operation. In this study, a two-phase flow Particle Tracking Velocimetry (PTV) algorithm was successfully used to characterize the spatial spray pattern and velocity field in two experimental showers (one low-flow and one high-flow). In addition, the airborne bacteria present in the shower over nearly 5 months of controlled operation was determined for both showers. The results indicate that the droplet velocity out of the low-flow showerhead (which had fewer orifices) was significantly higher than that out of the high-flow showerhead resulting in a higher aerosol number concentration in the low-flow shower and more consistent wetting of the shower wall. Both showerheads generated droplets in the respirable range and genera of potential health concern were observed in the shower aerosols measured both prior to and following shower operation. The study provides one of the first visualizations of droplet spray patterns in residential showers and provides insight into the airborne bacteria present in showers.ViewShow abstract... Los posibles ATGs se identificaron traduciendo los ESTs con ExPASy. Para verificar el tamaño de los cDNAs clonados, los plásmidos extraídos según el método de lisis alcalina (Zhou et al., 1990) se digirieron con la enzima BsrGI (Cat. No. R0575S, BioLabs) para escindir los cDNAs del vector pDONR222 donde se encuentran clonados (Martínez-Hernández et al., 2010). ...IDENTIFICACIÓN DE cDNAs DE Agave tequilana Weber Var. azul SIMILARES A FUROSTANOL GLICÓSIDO 26-O-β-GLUCOSIDASASArticleFull-text availableDec 2019REV FITOTEC MEX Zurisadai Monroy Aida Martinez-HernandezLa furostanol glicósido 26-O-β-glucosidasa (F26G) participa en el último paso de la biosíntesis de saponinas esteroidales, lo que escinde la glucosa unida al hidroxilo del C26 del furostanol glucósido para permitir la formación del espirostano. A pesar de la existencia de numerosos estudios que muestran la diversidad estructural y actividad biológica de las saponinas de plantas, así como su reconocido potencial biotecnológico y farmacológico, pocas enzimas participantes en la ruta de biosíntesis de las saponinas esteroidales han sido estudiadas y sólo una enzima nativa tipo F26G ha sido purificada y caracterizada funcionalmente. En este estudio, mediante búsqueda por BLASTX en una base de datos de Agave tequilana Weber var. azul (Agave DB), se identificaron 46 ESTs (Expressed Sequence Tags) de DNA complementario (cDNAs) similares a la F26G de Asparagus officinalis, la mayoría provienen de cDNAs clonados a partir de tejidos florales (anteras y ovarios) o raíz; ambos órganos son productores de saponinas. Entre ellos, se identificaron seis ESTs cuyo alineamiento indica que representan a tres cDNAs diferentes entre sí. Estos ESTs se registraron en la base de datos dbEST de NCBI (National Center for Biotechnology Information), el cual fue el primer registro de secuencias nativas de cDNAs potencialmente codificantes similares a F26G en Agave. El tamaño de los cDNAs clonados y su alineamiento hacia el lado 5’ de las F26G conocidas sugieren que son cDNAs completos. Se diseñaron iniciadores diferenciales específicos para cada tipo de cDNA y se analizó por RT-PCR su expresión en tejidos vegetativos de A. tequilana. Se establecieron protocolos de PCR cuantitativo (qPCR) para estudios posteriores de regulación de su expresión génica. La información aquí reportada servirá como base para estudiar la función, regulación y actividad enzimática de las enzimas clonadas de Agave similares a F26G.ViewShow abstract... ng mL -1 , estos resultados concuerdan con los reportados por Oslinger et al. (2006); Piedrahíta et al. (2008) y Escobar et al. (2014), quienes aplicaron este método para extraer eficientemente ácidos nucleicos |AN|. Por otro lado, el método de TENS sobre el tejido sanguíneo produjo un ADN entre 1667.7 a 1962.98 ng mL -1 , según reportes de Zhou et al. (1990) mencionan que este método no es muy eficiente para la extracción genómica en tejido animal (Cuadro 1). La ultrasensibilidad de detección por la PCR anidada permitió determinar qué; del total de las muestras analizadas, el 13.46% de las garrapatas Riphicephalus (Boophilus) microplus fueron positivas para A. marginale, de estos el 22.58% de la población pertenecen a las muestra extraídas de las haciendas y fincas; el 77.42% corresponden a las extraídas de la EMCQ. ...Prevalencia y detección por PCR anidada de Anaplasma marginale en bovinos y garrapatas en la zona central del Litoral ecuatorianoArticleSep 2015 Ariel Escobar Edgar Rodolfo Pinargote MendozaHelen Carranza PatiñoPatricio VillarrealLa bacteria que provoca la anaplasmosis en bovinos se conoce como Anaplasma marginale, es potencialmente transmitida de forma biológica por garrapatas, moscas y fómites o mediante sangre infectada como consecuencia del uso incorrecto de herramientas quirúrgicas. Hasta la fecha, Ecuador carece de estudios actualizados sobre procedimientos eficientes para el diagnóstico específico y, erradicación de A. marginale a través del control de estos insectos, por lo que resulta de gran importancia desarrollar e implementar trabajos relacionados con el uso de esta herramienta molecular, para la detección de la bacteria en vectores de transmisión que provocan la enfermedad en bovinos. En este trabajo, se extrajo ADN eficazmente con el método de Salting Out. Un total de 255 muestras fueron analizadas por PCR anidada, distribuidas del siguiente modo|108| Riphicephalus (Boophilus) microplus, |85| bovinos, de estos el 13.46% y 85.48% dieron positivas para la rickettsia, las muestras de Amblyomma spp. |62| todas fueron negativas. El índice de concordancia Kappa |total de bovinos infestados frente a Riphicephalus (Boophilus) microplus|, no fue significativo (0.28). Subsiguientemente, se determinó por χ2 (p=0.66) que la presencia o ausencia de la enfermedad es independiente del lugar de donde proviene el bovino.ViewShow abstract... For all the 14 isolates, plasmid DNA isolation was carried out following the TENS protocol (Zhou et al., 1990;Roig and Amaro, 2009). Samples were subjected to electrophoresis on horizontal 0.7% agarose gel at 100v for 2 hours and the DNA visualized with an ultraviolet transilluminator (VVP Inc.) after staining with 0.05% ethidium bromide. ...STUDIES ON EFFECTS OF BARK EXTRACTS OF Azadirachtaindica A. (JUSS) ON MULTIDRUG RESISTANT Salmonella TyphiArticleFull-text availableFeb 2016 Bobai Mathew Harriet UgbokoMathewN. *1DeThis investigation was aimed at determining the antimicrobial activity of extracts of stem bark ofA. indica A. Juss (neem) on some multidrug resistant Salmonella Typhi isolates obtained from patientssuffering from complications of typhoid fever. Preliminary phytochemical and spectrophotometricanalysis of acetone and ethanol bark extracts of A. indica revealed the presence of tannins, alkaloids,flavonoids, glycosides, saponins, ketones, phenolic compounds, carboxylic acids, aromatic compounds,amides, proteins, aldehydes and aliphatic compounds. At concentrations of 25-400 mg/ml, all the 14isolates were susceptible to the acetone and ethanol bark extracts of the A.indica though the diameters ofzones produced by various isolates at different concentrations of acetone and ethanol extracts weresignificantly different (the acetone extract had 18-35mm, while ethanol extract had 15-31mm diameter)Studies on plasmid extraction showed that all of the isolates carried large molecular weight plasmid(above 12,000 kb).The antibiotic resistance pattern of the isolates against different antibiotics showedthat the isolates had high resistance to augmentin and cotrimoxazole (100%) followed by cefuroxime(92.9%), tetracycline 85.7%, amoxicillin 78.6%, cefixime 71.4%, ceftriaxone 64.3%, chloramphenicol64.3% and nalidixic acid 57.1%, and had low resistance to ciprofloxacin (35.7%), ofloxacin andnitrofurantoin (14.3%). The minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) values of the acetone and ethanol extracts against the isolates were in the range of50-100 mg/ml respectively. It may be concluded from the results of this study that the bark extracts ofAzadirachta indica A. (JUSS) may be used to treat typhoid fever preceded by further experiments.ViewShow abstract... TENS mini -prep method was adopted as described by Kado and Liu [17], Ojo and Oso [30], Zhou et al. [46] to extract plasmid DNA of the isolates in the original wastewater sample. ...POTENTIAL EXOELECTROGENIC BACTERIA SPECIES ISOLATED FROM PIGGERY WASTEWATER USED IN GENERATION OF BIOELECTRICITY AND WASTEWATER TREATMENTArticleFull-text availableMar 2017 Toochukwu Ekwutosi OgbulieAbstract. The ability of bacteria in anode chambers of microbial fuel cell (MFC) to transfer electrons from their respiratorychains to anode distinguishes it into mediator or mediator-less MFC. Two groups of 3 MFCs each were constructed with eitherpotassium permanganate as electron acceptor, or potassium ferricyanide. Electrodes used were carbon – carbon (CC), carbon –copper (CCu) and copper – copper (CuCu) in each group. The initial BOD and COD of the piggery wastewater were 420mg/L and1057mg/L respectively. After 25 days, coulombic efficiency recorded were 69%, 84%, 74%, 76%,72% and 5.10%, while CODremoval 65%, 51%, 47%, 83%, 48% and 49% for CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Maximum powerdensity (at Rext= 1000Ω) observed were 79.27mW/m, 156.32mW/m, 92.29mW/m, 60.94mW/m, 39.94mW/mand 14.21mW/mfor CCP, CCuP, CuCuP, CCF, CCuF and CuCuF respectively. Although Streptococcus sp.,Salmonella sp., Lactobacillus sp.,Escherichia coli, Proteus mirabilis, Enterobacter sp.,Pseudomonas sp.,Bacillus sp.,Micrococcus luteus,Corynebacterium sp.,Shigella sp.and Aeromonas sp. were biochemically identified before treatment of wastewater, but Pseudomonas sp.,Escherichiacoli, Shigella sp.and Aeromonas sp. did not persist after treatment. Molecular analysis confirmed the absence of Clostridiumbotulinum, Aeromonas hydrophila, Clostridium butyricum andRhodobacter ferrireducens, which are known exoelectrogens on thesurface of anodes. Plasmid profile revealed that Lactobacillus sp., Proteus mirabilis, Escherichia coli, Pseudomonas sp., Bacillussp., and Aeromonas sp. carried plasmids. Studies should be undertaken using these persistent bacteria in isolation to ascertain theirindividual capabilities, together with other cheaper, more environmentally friendly catholytes for better outputsViewShow abstract... General molecular biology techniques were followed (Sambrook et al., 1989). Plasmid DNA from Pseudomonas was purified by a rapid alkaline lysis (Zhou et al., 1990); plasmids from E. coli were purified using a boiling lysis method (Holmes and Quigley, 1981) or a commercial kit (Illustra plasmid Prep Mini Spin Kit, GE Healthcare). Undigested plasmids were separated in 0.8% agarose gels by electrophoresis (Sesma et al., 2000). ...Genes ptz and idi, Coding for Cytokinin Biosynthesis Enzymes, Are Essential for Tumorigenesis and In Planta Growth by P. syringae pv. savastanoi NCPPB 3335ArticleFull-text availableAug 2020Maite Añorga Adrián Pintado Cayo Ramos Jesús MurilloThe phytopathogenic bacterium Pseudomonas syringae pv. savastanoi elicits aerial tumors on olive plants and is also able to synthesize large amounts of auxins and cytokinins. The auxin indoleacetic acid was shown to be required for tumorigenesis, but there is only correlational evidence suggesting a role for cytokinins. The model strain NCPPB 3335 contains two plasmid-borne genes coding for cytokinin biosynthesis enzymes: ptz, for an isopentenyl transferase and idi, for an isopentenyl-diphosphate delta-isomerase. Phylogenetic analyses showed that carriage of ptz and idi is not strictly associated with tumorigenic bacteria, that both genes were linked when first acquired by P. syringae, and that a different allele of ptz has been independently acquired by P. syringae pv. savastanoi and closely related bacteria. We generated mutant derivatives of NCPPB 3335 cured of virulence plasmids or with site-specific deletions of genes ptz and/or idi and evaluated their virulence in lignified and micropropagated olive plants. Strains lacking ptz, idi, or both produced tumors with average volumes up to 29 times smaller and reached populations up to two orders of magnitude lower than those induced by strain NCPPB 3335; these phenotypes reverted by complementation with the cloned genes. Trans-zeatin was the most abundant cytokinin in culture filtrates of NCPPB 3335. Deletion of gene ptz abolished biosynthesis of trans-zeatin and dihydrozeatin, whereas a reduced but significant amount of isopentenyladenine was still detected in the medium, suggesting the existence of other genes contributing to cytokinin biosynthesis in P. syringae. Conversely, extracts from strains lacking gene idi contained significantly higher amounts of trans-zeatin than extracts from the wild-type strain but similar amounts of the other cytokinins. This suggests that Idi might promote tumorigenesis by ensuring the biosynthesis of the most active cytokinin forms, their correct balance in planta, or by regulating the expression of other virulence genes. Therefore, gene ptz, but not gene idi, is essential for the biosynthesis of high amounts of cytokinins in culture; however, both ptz and idi are individually essential for the adequate development of tumors on olive plants by Psv NCPPB 3335.ViewShow abstract... Alternate target genes include katE, sodC, and mdtM, which also have mutant alleles associated with enhanced resistance (Weiss et al. 2016). For each aerosol sample, DNA extraction can be performed with three replicate samples using standard methods (Zhou, Yang, and Jong 1990). Following isolation and purification, DNA from the replicates and time points for each aerosol sample can be prepared as barcoded libraries and sequenced on an Illumina HiSeq 2000. ...Assays and Enumeration of Bioaerosols-Traditional Approaches to Modern PracticesArticleFull-text availableFeb 2020AEROSOL SCI TECH Ronald Lacey Hyoungmook PakAlexandra Koustova Maria D KingIn recent years, much attention has been drawn to the exposure to bioaerosols in both occupational and indoor environments due to adverse effects on human health. Exposure to these agents may cause infectious diseases, allergic diseases, acute toxic effects, respiratory diseases, neurological effects, and cancer.Bioaerosols play a significant role in air quality studies, as well as in industrial and agricultural regulations. There is a diversity of sources for bioaerosol exposures, which include occupational activities such as waste disposal, sorting and composting, agricultural and food processing, livestock production and handling, healthcare, and fungal growth following flooding. Bioaerosol monitoring includes the measurement of viable (culturable and nonculturable) and nonviable microorganisms in both indoor and outdoor environments. The development of standardized methods for the detection and quantitation of bioaerosols has become an important issue. Technologies, including microbial plating, physical-chemical assays, and molecular techniques, have been adapted for the assessment of collected bioaerosol particles. In this review, we will discuss traditional and modern assays that have been applied in bioaerosol enumeration based on culturability, optical properties, immunoreactions, and nucleic acid amplification and sequencing of biological particles. The analysis of additional characteristics of bioaerosols including microbiome and antimicrobial resistance is also discussed. Finally, concluding thoughts are offered regarding the challenges and perspectives in the field.Copyright © 2020 American Association for Aerosol ResearchViewShow abstract... Plasmid profiling of the four gram-negative isolates were carried out, by following the TENS-Mini Prep method Zhou et al., 1990. Plasmid DNA isolation (TENS prep) 1. Overnight culture of 1.5 ml was dispensed into a microfuge tube 2. The deposit was spun for 1 min at 10rpm to pellet the cells 3. The supernatant was discarded, and 150ul of media was left in the tube 4. It was vortexed to re-suspend the cells 5. TENS buffer of 300μl was added to the vortexed cells 6. ...Microbiological Assessment of Commercial Yogurt Sold in Ota Metropolis, Ogun State, Nigeria.ArticleFull-text availableDec 2018 Olugbenga TaiwoR.O. Afolabi Oranusi Solomon Ige OjoTen authorized Yogurt products purchased within Ota metropolis, Ogun State of Nigeria were subjected to pH, Total Bacterial Count (TBC) and Total Fungal Count (TFC) analysis. pH values were in the range of 4.05 to 5.50, the TBC and TFC values ranged between 1.0 x 103 - 5.0 x 105 cfu/ml and 1.0 x 103 - 5.0 x 105 cfu/ml respectively. Eleven bacterial isolates were detected in the yogurt samples. Lactobacillus spp. and Bacillus spp. constituted 16% of the total microbial load, Corynebacterium spp., Klebsiela spp., Staphylococcus spp., and Pseudomonas spp. constituted 8% while Proteus spp., Micrococcus spp., Shigella spp., Listeria spp., and Streptococcus spp.constituted 4%. Fungal isolates obtained were Mucor spp. (22%), Geotrichum spp. (17%), Montospora spp. (11%), while Aspergillus spp., Rhizopus spp., and Fusanrium spp. constituted 6%.The antimicrobial susceptibility test showed that the isolates exhibited susceptible toCiprofloxacin and Ofloxacin and resisted Nitrofurantoin, Augumentin, Cefixime, Ceufuroxime, Gentamicin and Ceftazidime. The isolates were plasmid encoded, with size range of 20,000- 40,000 Kilo base pairs. Result show no significant difference within the bacteria isolates (P 0.05), while the fungi isolates showed significant difference (P 0.05). Significant difference also occurred between the bacteria and the fungi isolate (P 0.05).ViewShow abstract... DNA Extraction. The Alkaline Lysis method was used to extract DNA from all the samples (Zhou et al., 1990). The DNA concentration was determined by measuring optical Splitting-washing Fabrication room density at 260 nm (OD 260 ) using a spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del.). ...Monitoring of Pathogenic Bioaerosols in Beef Slaughter Facilities Based on Air Sampling and Airflow ModelingArticleJan 2019Samuel H Beck Alejandro Castillo Kerry A Kinney Maria D KingHighlights Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).Significant bioaerosol concentrations were detected in beef slaughter facilities Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).Airflow was modeled in beef facilities and potential pathogens were tracked Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).Results indicate transport of bioaerosols toward chiller with final food product Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).New ventilation designs indicate that plant sanitation can be improved Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).Additional studies are required to verify effectiveness of new ventilation Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).Abstract. and Shiga toxin producing (STEC) have long been recognized as pathogens of concern in meat products due to the prevalence of these microorganisms in the gastrointestinal tract and hide of livestock. Bacterial ingestion due to contaminated food products causes a great economic burden from the hospitalization and death of those who become infected. Recently, aerosolized bacteria have been recognized as a threat to human health and shelf life of food. In beef processing facilities, the majority of harmful bacteria are introduced by the cattle. Heating, ventilation, and air conditioning (HVAC) systems can harbor and transport these microscopic organisms. Salmonella and STEC cause 78 billion dollars lost every year due to contaminated food. During the harvesting process, these pathogens may become aerosolized from the carcasses by various mechanisms, including worker activity and airflow from HVAC systems. Although bacteria are robust creatures, environmental conditions including ventilation can be manipulated to disrupt their proliferation. In this study, one rural and one small beef facility were examined. High air volume wetted wall cyclone bioaerosol samplers capable of collecting and concentrating bioaerosols in a liquid effluent were used during the entire processing at bleeding, de-limbing, de-hiding, washing, and chiller locations. Bioaerosols were analyzed using microbial plating, quantitative Polymerase Chain Reaction, and microbiome analysis. Total bacteria counts, STEC, and concentrations were enumerated in the air and critical areas were identified. and STEC were found to increase with each passing day in the facility, as well, total counts and STEC increased between morning and afternoon phases of processing. Significant differences in total counts and temperature were found at different locations in the facilities. Blueprints were obtained from the examined facilities and the cattle processing floors were modeled using computational fluid dynamics. The airflow created from the HVAC systems was found to have a significant effect on the spread of bioaerosols. Similarities were found between the collected concentrations of bioaerosols and particle traces in the modeled facilities. Finally, new ventilation models were generated to significantly increase the sanitation of the beef slaughtering process. Keywords: Airflow pattern, Beef processing facilities, Bioaerosols, Computational fluid dynamics modeling (CFD), Displacement ventilation, Wetted Wall Cyclone (WWC).ViewShow abstract... DNA Extraction. The Alkaline Lysis method was used to extract DNA from all the samples (Zhou et al., 1990). The DNA concentration was determined by measuring optical Splitting-washing Fabrication room density at 260 nm (OD 260 ) using a spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del.). ...Monitoring of Pathogenic Bioaerosols in Beef Slaughter Facilities Based on Air Sampling and Airflow ModelingArticleFull-text availableDec 2019APPL ENG AGRICSamuel Back Alejandro Castillo Kerry A Kinney Maria D Kingand Shiga toxin producing (STEC) have long been recognized as pathogens of concern in meat products due to the prevalence of these microorganisms in the gastrointestinal tract and hide of livestock. Bacterial ingestion due to contaminated food products causes a great economic burden from the hospitalization and death of those who become infected. Recently, aerosolized bacteria have been recognized as a threat to human health and shelf life of food. In beef processing facilities, the majority of harmful bacteria are introduced by the cattle. Heating, ventilation, and air conditioning (HVAC) systems can harbor and transport these microscopic organisms. Salmonella and STEC cause 78 billion dollars lost every year due to contaminated food. During the harvesting process, these pathogens may become aerosolized from the carcasses by various mechanisms, including worker activity and airflow from HVAC systems. Although bacteria are robust creatures, environmental conditions including ventilation can be manipulated to disrupt their proliferation. In this study, one rural and one small beef facility were examined. High air volume wetted wall cyclone bioaerosol samplers capable of collecting and concentrating bioaerosols in a liquid effluent were used during the entire processing at bleeding, de-limbing, de-hiding, washing, and chiller locations. Bioaerosols were analyzed using microbial plating, quantitative Polymerase Chain Reaction, and microbiome analysis. Total bacteria counts, STEC, and concentrations were enumerated in the air and critical areas were identified. and STEC were found to increase with each passing day in the facility, as well, total counts and STEC increased between morning and afternoon phases of processing. Significant differences in total counts and temperature were found at different locations in the facilities. Blueprints were obtained from the examined facilities and the cattle processing floors were modeled using computational fluid dynamics. The airflow created from the HVAC systems was found to have a significant effect on the spread of bioaerosols. Similarities were found between the collected concentrations of bioaerosols and particle traces in the modeled facilities. Finally, new ventilation models were generated to significantly increase the sanitation of the beef slaughtering process.ViewShow abstractSequence-specific Polymerase Chain-reaction Markers Derived from Randomly Amplified Polymorphic DNA Markers for Fingerprinting Grape (Vitis) RootstocksArticleJan 1996J AM SOC HORTIC SCIHong XuDiane J. WilsonS. ArulsekarAlan T BakalinskyRandomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.ViewShow abstractConsensus PCR protocols for the detection of amphibian herpesviruses ( Batrachovirus )ArticleAug 2020J VET DIAGN INVESTMatthias Licheri Francesco C OriggiAmphibians have been disappearing at an unprecedented rate worldwide. Among the proposed contributing factors are infectious diseases. Investigations have focused mainly on ranavirus and chytrids; however, additional agents may be relevant stressors. Two novel batrachoviruses have been discovered (ranid herpesvirus 3 [RaHV-3] and bufonid herpesvirus 1 [BfHV-1]). Their clinical role is still to be clarified; however, both have been associated with obvious skin lesions in their respective hosts. Herein we present 2 consensus PCR protocols that can be used to detect all of the known and, possibly, yet to be discovered batrachoviruses. We targeted a 200 nt long, highly conserved region of the DNA terminase gene. We established a sensitive protocol, which can detect both European batrachoviruses (European batrachovirus PCR protocol; RaHV-3 and BfHV-1) and a panbatrachovirus PCR protocol detecting all known batrachoviruses, including ranid herpesvirus 1 and 2 (RaHV-1, -2). The limit of detection (LOD) for the European batrachovirus protocol was 10 ¹ copies of RaHV-3 and 10 ² copies of BfHV-1 per reaction. The panbatrachovirus protocol could detect all known batrachoviruses with LODs of 10 ³ (RaHV-3, BfHV-1, RaHV-1) to 10 ⁴ copies (RaHV-2) per reaction. These novel detection tools can be used as a first line of detection when herpesviral infection in amphibians is suspected, followed by additional PCRs with herpesvirus-specific primers in the case of known viral species, or sequencing as in the case of novel batrachoviruses.ViewShow abstractUse of the Different Polyacrylamide Gel Electrophoresis (PAGE) Methods in the Characterization of Human Pathogenic Staphylococcus aureus StrainsArticleApr 2010CHEM-ASIAN J Ismet BerberNegmetullah AlanSuat Ekin Harun OnluIn the study, a total of 41 strains of Staphylococcus aureus including 38 clinical isolates and 3 reference strains were characterized according to biotyping, plasmid profiles and the numerical analysis of the protein profiles. The antibiogram results showed that the isolates were resistant against the tested antibiotics, except for vancomycin. Furthermore, plasmid profile analysis confirmed that the strains consisted of only one plasmid. Present findings indicated that the whole-cell and extracellular protein profiles obtained by using SDS-PAGE methods to be good typing tool for the differentiation of S. aureus strains at the species level, not strain level. However, Native-PAGE of whole-cell proteins was the most reliable and rapid method for differentiation between MRSA and ordinary S. aureus strains comparing to other applying PAGE techniques. In addition, it was determined that the same clone was responsible for most cases of MRSA and other S. aureus epidemic among surveyed hospitals. In conclusion, this study supposed that together application at least two different polyacrylamide gel electrophoresis (PAGE) techniques can be offer accurate and an effective approach to the investigation of taxonomic relationships within human pathogenic S. aureus strains.ViewShow abstractPseudomonas savastanoi pv. mandevillae pv. nov., a clonal pathogen causing an emerging, devastating disease of the ornamental plant Mandevilla sppArticleJan 2021PHYTOPATHOLOGY Eloy Caballo Ponce Alba Moreno Pérez Adrián Pintado Cayo RamosCommercial production of the ornamental plant dipladenia (Mandevilla spp.) is threatened by dipladenia leaf and stem spot disease, caused by the bacterium Pseudomonas savastanoi. P. savastanoi includes four pathovars of woody hosts differentiated by a characteristic host range in olive, oleander, ash and broom plants. However, isolates from dipladenia have not been ascribed to any particular lineage or P. savastanoi pathovar. Here we report that isolates from dipladenia represent a distinct, clonal lineage. First, dipladenia isolates display very similar plasmid profiles, including a plasmid encoding the iaaM gene for biosynthesis of indole-3-acetic acid. Second, multilocus sequence analysis and core-genome single-nucleotide-polymorphisms phylogenies showed a monophyletic origin for dipladenia isolates, which cluster with isolates from oleander (pathovar nerii) in a distinct clade well separated from other P. savastanoi strains. Metabolic profiling and cross-pathogenicity tests in olive, oleander, ash, broom and dipladenia clearly distinguished dipladenia isolates from the four P. savastanoi pathovars. Comparative genomics of the draft genome sequence of the dipladenia strain Ph3 with the other four pathovars showed that Ph3 encodes very few strain-specific genes, and a similar set of virulence genes to pv. nerii, including its repertoire of type III secretion system effectors. However, hierarchical clustering based on the catalogue of effectors and their allelic variants clearly separated Ph3 from pv. nerii strains. Based on their distinctive pathogenicity profile, we propose a de novo pathovar for P. savastanoi isolates from dipladenia, P. savastanoi pv. mandevillae pv. nov., for which strain Ph3 (CFBP 8832 PT ) has been designated as the pathotype strain.ViewShow abstractTransformation of Nematodes by MicroinjectionChapterFull-text availableJan 1999 Syeda HashmiGhazala Hashmi Randy GauglerThe free-living nematode, Caenorhabditis elegans, has been an important model system in the study of developmental and cell biology. Significant advances in mapping and sequencing the C. elegans genome have aided our understanding of fundamental biological processes. Development of an efficient method for gene transfer has been a key tool accelerating C. elegans research advances (Kimble et al., 1982; Stinchcomb et al., 1985., Fire, 1986; Mello et al, 1991). Integrative transformation in C. elegans is reproducibly achieved after microinjecting DNA directly into maturing oocyte nuclei (Fire, 1986). Heritable extrachromosomal DNA transformation in C. elegans was first described by Stinchcomb et al. (1985) after microinjecting DNA into the gonad cytoplasm. DNA molecules injected into the cytoplasm of the C. elegans hermaphrodite gonad undergo a transient period of reactivity. This results in the formation of large heritable extrachromosomal structures that experience very little further rearrangement (Mello et al., 1991). Germ cell nuclei in C. elegans develop initially in a syncytium (a multinucleate mass of protoplasm resulting from fusion of cells) and when cell membranes later envelop them, the exogenously added DNA is packaged into the oocyte. Transforming DNA is generally not integrated into the chromosomes, but rather is maintained as a concatamer (i.e. unite in a chain) of introduced sequences.ViewShow abstractCloning of Flavin Reductase into pET32a(+) Expression Vector Lacking the Thioredoxin A Tag to Study Solubility of EDTA Monooxygenase A in Overexpression SystemsArticleSiaw Hui WongThe formation of inclusion bodies is a major obstacle for getting efficient bacterial protein production in expression systems. The pET32a(+) expression vector from Novagen fuses protein of interest to thioredoxin A (TrxA) to help facilitate disulfide bond formation in the cytoplasm greatly reducing protein aggregates. Flavin oxidoreductase (Fre) is another thioredoxin enzyme and may present another option. An attempt was made to clone the PCR amplified fre gene into pET32a(+) that had the thioredoxin A (TrxA) tag removed. Since it is unknown how the redox state of thioredoxin enzymes are able to improve protein solubility we wanted to compare expression of the highly insoluble protein EDTA monooxygenase A (EmoA) as a fusion protein to Fre and as independent co-expression. The project was delayed at the cloning step due to the difficulty of working with the NdeI restriction enzyme. It was found that a ligation condition with a low amount of T4 DNA ligase improve the poor ligation efficiency of NdeI digested fragments. A re-circularized vector with the TrxA tag removed was made as well as three potential clones that may contain the Fre insert. Attempts to clone EDTA monooxygenase A into the new vector resulted in no clones. _______________________________________________________________ Protein expression using bacterial systems have many advantages, such as rapid growth rate, ease of manipulation, and the ability to produce milligram quantities of proteins. A major caveat of the system is the formation of highly insoluble inclusion bodies in the cytoplasm resulting in non-functional protein products. To overcome this, proteins are expressed fused to a redox enzyme, such as thioredoxin A (TrxA). Thioredoxin are small enzymes that undergo redox reactions through the reversible oxidation of two cysteine thiol groups to a disulphide resulting in the transfer of two electrons and two protons. Protein fusion with TrxA lead to increased disulfide bond formation, decreasing the formation of proteins aggregates. Many have used this strategy, for example the catalytic domain of human 11-β-hydroxysteroid dehydrogenase type 1(11β-HSD1), a membrane bound protein, when expressed fused to TrxA was found to be functionally active (3). Banerjee et al. also reported the ability to produce functionally active scorpion neurotoxin Lqq-V in Escherichia coli. Both of these proteins contain many disulfide bonds in their structure and if expressed without TrxA would have formed highly insoluble aggregates preventing any expression studies to be carried out. The mechanism as to how thioredoxin stimulate disulfide bond formation between two protein cysteine residues within the reducing environment of bacterial cytoplasm has been studied and elucidated; however, the extent to which protein solubility is improved is yet unknown. It is known in E. coli that a loss of function mutation in thioredoxin reductase (TrxB) no longer permits TrxA to carry out its reducing reactions leading to a less reduced environment in the cytpolasm. The commercially available pET32a(+) vector from Novagen permits genes of interest to be fused to trxA for high levels of expression with good solubility. Flavin reductase (Fre) is another redox enzyme with similar catalytic reduction abilities as TrxA. In this study, we attempt to clone fre into an expression vector and determine if it is able to increase the solubility of overexpressed proteins. EDTA monooxygenase A (EmoA), a highly insoluble protein when overexpressed, was chosen to investigate the effects of the redox enzyme in reducing the formation of protein aggregates in overexpression system (2). More specifically, we want to determine if the increase in protein solubility is due to the reductase enzyme activity or due to fusion protein properties. A construct of pET32a(+) vector with its trxA tag removed was made. Attempts were made to clone fre into the recircularized expression vector resulting in six potential clones.ViewShow abstractRapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion ConstructsArticleJun 2017JoVEScott E. GoldZahi Paz María D. García-Pedrajas Anthony E GlennPrecise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5 and 3 flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.ViewShow abstractA study of the myosin heavy chain gene family in the carpThesisJan 1994Steven James EnnionSkeletal muscle has a striking potential for plasticity with an inherent ability to adapt to altered functional demands. The expression of various isoforms of the myosin heavy chain (MyoHC) plays a central role in facilitating these adaptive changes by conferring changes in contractile characteristics. In contrast to mammals, very little is known about the MyoHC isoforms and their genes in fish. This study aimed to characterise the family of MyoHC isogenes in the carp (Cyprinus carpio). Using genomic clone analysis and 3 RACE-PCR, the 3 untranslated regions of seven separate carp MyoHC isogenes (arbitrarily named types 1 to 7) were isolated and used to characterise the expression patterns of individual MyoHC isoforms. Northern blot analysis demonstrated that the carp MyoHC gene family is developmentally regulated and that their expression is also determined by environmental temperature. Two isoforms, types 1 and 5, are expressed in both adult and immature carp and three, types 2, 3 and 4, are expressed exclusively in immature carp. In situ hybridisation localised the expression of the type 2 MyoHC isoform to the developing pink muscle fibre layer in fry and 12 month old carp and demonstrated that distinct isoforms of the MyoHC are expressed in the red and white muscle fibres types. Types 6 and 7 MyoHC isoforms were shown to be expressed exclusively in the white muscle fibres of adult carp which had been acclimated to a warm (28°C) temperature. The expression of the type 7 MyoHC gene was localised, by in situ hybridisation, to the small diameter (10-25?m) white muscle fibres which are thought to be involved in fibre hyperplasia. The size of this gene at the genomic level was shown to be half the size of mammalian MyoHC isoforms and this difference in size was attributed to shorter introns.ViewShow abstractClass I integrons containing a dhfrI trimethoprim resistance gene cassette in aquatic Acinetobacter spp.ArticleJan 2000FEMS MICROBIOL LETTA PetersenViewStudy of the virulence in wild-type strains of Erwinia amylovora devoid of the plasmid pEA29ArticleJun 2008 Pablo Llop María M. López Jordi Cabrefiga Emilio MontesinosThe recent discovery of Spanish strains of Erwinia amylovora that do not harbor the plasmid pEA29, opens new possibilities to study the role of this plasmid in virulence of E. amylovora. A screening of the plasmid content of strains from international collections and isolates from Spain has been performed and eight strains lacking the pEA29 have been found so far (three from Spain, two from Serbia-Montenegro, two from Belgium and one from the United Kingdom). These strains were compared to reference strains of E. amylovora. The results showed very similar biochemical and molecular characteristics; however, four of the strains carried a plasmid of higher size (circa 70 kb), named pEI70, as demonstrated by restriction analyses and hybridization assays. The symptoms produced by these strains in nature were similar to that of strains that carry pEA29, and these results have led to study the virulence level of these strains and the possible relationship with their plasmid content because of the described effect of pEA29 on this feature. The virulence analyses after inoculation of immature pear showed the existence of differential level of virulence depending on the strain, but all the strains without the pEA29 increased their virulence after the introduction of this plasmid, confirming its effect on the virulence of E. amylovora.ViewShow abstractSTUDY ON THE USE OF MICROBIAL FUEL CELL AS WASTE MANAGEMENT OPTION TO GENERATE ELECTRICITY FROM PIGGERY WASTEWATERArticleFull-text availableMay 2017 Toochukwu Ekwutosi OgbulieAbstract. Microbial fuel cells (MFCs) were constructed to demonstrate the feasibility of generating bioelectricity from piggerywastewater. Exoelectrogens in the wastewater were harnessed and 0.1M potassium permanganate (KMnO4) solution served aselectron acceptor. Three units of 2-chambers MFCs with carbon – carbon (CC), carbon – copper (CCu) and copper-copper (CuCu)electrodes were constructed. Using piggery wastewater of BOD, 420mg/L and COD, 1057mg/L, the highest open circuit voltage(OCV) recorded were 969.6mV, 1228.5mV and 1338.5mV for CC, CCu and CuCu respectively. The voltage recorded across theMFCs was observed to decrease with decreasing external resistance. The highest power density (at Rext= 1000Ω) were 79.27mW/m(105.7mA/m), 156.32mW/m(148.4mA/m) and 92.29mW/m(114.0mA/m) for CC, CCu and CuCu respectively. Generally,power density increased with decreasing external resistance across each MFC until 200Ωbeyond which it decreased. After 25daysoperation of the MFCs, the coulombic efficiency of the MFCs were 69%, 84% and 74%, while COD removal were 65%, 51% and47% for CC, CCu and CuCu respectively. Moreover, carbon – carbon electrodes mix was found to be better in generation ofelectricity and wastewater treatment than copper – copper electrodes or their combinations. Pre and post bacteria isolation andidentification revealed the presence of Lactobacillus sp., Corynebacterium sp., Streptococcus sp., Proteus mirabilis, Enterobactersp., Escherichia coli, Pseudomonas sp., Bacillus sp., Aeromonas sp., Micrococcus luteus,Corynebacterium sp.and Salmonella sp.in the test piggery wastewater. This study therefore paved way for further optimization and scale up for better harvest of energy andwastewater treatmentViewShow abstractEpiphytic Fitness of Pseudomonas syringae pv. syringae on Mango Trees is Increased by 62-Kb PlasmidsChapterJan 2003 Eva Arrebola Francisco M. CazorlaC AbadAntonio de VicenteIn this work, we have studied the relationship between presence of indigenous plasmids and copper- and ultraviolet light-resistance in Pseudomonas syringae pv. syringae strains isolated from mango trees. In such strains, plasmids of 62 kb showed cross-hybridisation with specific sequences of the copper-resistance operon copABCD and the UV-resistance determinant rulAB. Copper-resistance was evaluated by determining the minimal inhibitory concentration (MIC) of copper sulphate, as well as analysing the increase of copper-resistant (Cur) bacteria in commercial field plots under copper treatments during three years. UV-resistance was evaluated by performing survival curves of P. s. pv. syringae cells exposed to doses of B+A UV-fractions, as well as the persistence of P. s. pv. syringae strains under field conditions studied on mango leaves directly exposed to solar radiation. Survival curves showed that strains harbouring 62-kb plasmids had a higher survival ability under both conditions. The role of the indigenous plasmids of 62 kb in copper- and UV-resistance was confirmed by evaluating survival of transconjugants obtained by mating assays. The most abundant 62-kb plasmid (62.1) carried copper- and UV-resistance determinants homologous to copABCD and rulAB genes, and was shown to be conjugative and very stable. These results strongly suggest an important contribution of 62-kb plasmids to increasing the epiphytic fitness of P. s. pv. syringae on mango plant surfaces.ViewShow abstract2. *OJO-OMONIYI O.A. (2013). Biodegradation of Synthetic Detergents In: Biodegradation-Life of Science (Ed.) Dr. Rolando Chamy and Francisa Rosenkranz. Publ. InTech Open Science Europe. www.intechopen.com/books/biodegration ISBN: 978-953-51-11 54-2 http: 11dx doi. Org/10.5772/56461. Pp. 229 -250. Croatia, Europe.http://cdn.intechopen.com/pdfs/45110/InTech-Biodegradation_of_synthetic_detergents.pdfBookJan 2013 Olusola Abayomi Ojo--OmoniyiViewStudy on the use of microbial fuel cell as waste management option to generate electricity from piggery wastewaterArticleFull-text availableJan 2017 Uchechukwu Ezeji Henry Uzoma Anuforo Ogbulie Te Campbell AkujobiMicrobial fuel cells (MFCs) were constructed to demonstrate the feasibility of generating bioelectricity from piggery wastewater. Exoelectrogens in the wastewater were harnessed and 0.1M potassium permanganate (KMnO4) solution served as electron acceptor. Three units of 2-chambers MFCs with carbon - carbon (CC), carbon - copper (CCu) and copper-copper (CuCu) electrodes were constructed. Using piggery wastewater of BOD, 420mg/L and COD, 1057mg/L, the highest open circuit voltage (OCV) recorded were 969.6mV, 1228.5mV and 1338.5mV for CC, CCu and CuCu respectively. The voltage recorded across the MFCs was observed to decrease with decreasing external resistance. The highest power density (at Rext = 1000ω) were 79.27mW/m²(105.7mA/m²), 156.32mW/m² (148.4mA/m²) and 92.29mW/m² (114.0mA/m²) for CC, CCu and CuCu respectively. Generally, power density increased with decreasing external resistance across each MFC until 200ω beyond which it decreased. After 25days operation of the MFCs, the coulombic efficiency of the MFCs were 69%, 84% and 74%, while COD removal were 65%, 51% and 47% for CC, CCu and CuCu respectively. Moreover, carbon - carbon electrodes mix was found to be better in generation of electricity and wastewater treatment than copper - copper electrodes or their combinations. Pre and post bacteria isolation and identification revealed the presence of Lactobacillus sp., Corynebacterium sp., Streptococcus sp., Proteus mirabilis, Enterobacter sp., Escherichia coli, Pseudomonas sp., Bacillus sp., Aeromonas sp., Micrococcus luteus, Corynebacterium sp. And Salmonella Sp. In the test piggery wastewater. This study therefore paved way for further optimization and scale up for better harvest of energy and wastewater treatment.ViewShow abstractGenetic Transformation of Pimpinella anisum (Anise)ChapterJan 1999 Barry V. CharlwoodKhaled M. S. A. SalemPimpinella anisum L. (Apiaceae), known as anise or aniseed, is native to Egypt, Asia Minor, and Greece, but is now cultivated worldwide, particularly in Spain, France, Turkey, Syria, Chile, China, and USA.ViewShow abstractShow moreResearchGate has not been able to resolve any references for this publication.RecommendationsDiscover moreProjectexosomes Ambrose JongView projectArticle Midi-Prep isolation of plasmid DNA in less than two hours for sequencing, subcloning and hybridiza...November 1989 · BioTechniques Kathy LiszewskiV Kumar John P AtkinsonRead moreLooking for the full-text?You can request the full-text of this article directly from the authors on ResearchGate.Request full-textAlready a member? 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