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  • Biotechnology & Synthetic Biology  (41)
  • Oxford University Press  (41)
  • 1
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    OmpF of Pectobacterium carotovorum subsp. carotovorum Pcc3 is required for carocin D sensitivity (2016)
    Oxford University Press
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    Publication Date: 2016-12-23
    Description: Carocin D is a bacteriocin produced by Pectobacterium carotovorum subsp. carotovorum Pcc21. Carocin D inhibits the growth of P . carotovorum subsp. carotovorum and closely related strains. Pectobacterium carotovorum subsp. carotovorum is a causative bacterium for soft rot disease and leads to severe economic losses. Bacteriocins recognize and interact with a specific membrane protein of target bacteria as a receptor. To identify the receptor responsible for carocin D recognition, mutants that underwent a phenotypic change from carocin D sensitivity to carocin D insensitivity were screened. Based on Tn 5 insertions, carocin D sensitivity was dependent on expression of the outer membrane protein OmpF. The insensitivity of the mutant (Pcc3MR) to carocin D was complemented with ompF from carocin D-sensitive strains, not from carocin D-resistant strains. The selectivity between sensitive and resistant strains could be attributed to variation in OmpFs in the cell-surface-exposed regions. Based on sequence analysis and complementation assays, it appears that carocin D uses OmpF as a receptor and is translocated by the TonB system. According to previously reported translocation mechanisms of colicins, OmpF works along with the TolA system rather than the TonB system. Therefore, the current findings suggest that carocin D is imported by a unique colicin-like bacteriocin translocation system.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    The enhanced immune responses induced by Salmonella enteritidis ghosts loaded with Neisseria gonorrhoeae porB against Salmonella in mice (2016)
    Oxford University Press
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    Publication Date: 2016-12-16
    Description: Human health has been seriously endangered by highly prevalent salmonellosis and multidrug-resistant Salmonella strains. Current vaccines suffer from variable immune-protective effects, so more effective ones are needed to control Salmonella infection . Bacterial ghosts have been produced by the expression of lysis gene E from bacteriophage PhiX174 and can be filled with considerable exogenous substances such as DNA or drugs as a novel platform. In this study, Salmonella enteritidis (SE) ghosts were developed and loaded with Neisseria gonorrhoeae porin B (porB) to construct a novel inactive vaccine. Our new studies show that SE ghosts loaded with porB displayed increased production of pro-inflammatory cytokines (IL-1β, IL-6, IL-10 and IL-12p70) in bone marrow-derived dendritic cells (BMDCs), and elicited significantly higher specific systemic and mucosal immune responses to Salmonella than SE ghosts alone. In addition, the novel porB-loaded ghosts conferred higher protective effects on virulent Salmonella challenge. For the first time, we demonstrate that N. gonorrhoeae porB, as a novel adjuvant, can increase the immunogenicity of SE ghosts. Our studies suggested that Salmonella enteritidis ghosts loaded with Neisseria gonorrhoeae porin B might be a useful mucosal Salmonella vaccine candidate for practical use in the future.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 3
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    Expression of biologically active murine interleukin-18 in Lactococcus lactis (2016)
    Oxford University Press
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    Publication Date: 2016-12-12
    Description: The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900 . The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)- production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3–4 μg mL –1 and 0.6–0.7 ng mL –1 , respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 4
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    Enterotoxigenic Escherichia coli heat-stable toxin and heat-labile toxin toxoid fusion 3xSTaN12S-dmLT induces neutralizing anti-STa antibodies in subcutaneously immunized mice (2016)
    Oxford University Press
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    Publication Date: 2016-11-17
    Description: Enterotoxigenic Escherichia coli (ETEC) bacteria producing heat-stable toxin (STa) and/or heat-labile toxin (LT) are among top causes of children's diarrhea and travelers’ diarrhea. Currently no vaccines are available for ETEC associated diarrhea. A major challenge in developing ETEC vaccines is the inability to stimulate protective antibodies against the key STa toxin that is potently toxic and also poorly immunogenic. A recent study suggested toxoid fusion 3xSTa N12S -dmLT, which consists of a monomer LT toxoid (LT R192G/L211A ) and three copies of STa toxoid STa N12S , may represent an optimal immunogen inducing neutralizing antibodies against STa toxin [IAI 2014, 82(5):1823-32]. In this study, we immunized mice with this fusion protein following a different parenteral route and using different adjuvants to further characterize immunogenicity of this toxoid fusion. Data from this study showed that 3xSTa N12S -dmLT toxoid fusion induced neutralizing anti-STa antibodies in the mice following subcutaneous immunization, as effectively as in the mice under intraperitoneal route. Data also indicated that double mutant LT (dmLT) can be an effective adjuvant for this toxoid fusion in mice subcutaneous immunization. Results from this study affirmed that toxoid fusion 3xSTa N12S -dmLT induces neutralizing antibodies against STa toxin, suggesting this toxoid fusion is potentially a promising immunogen for ETEC vaccine development.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 5
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    Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion (2016)
    Oxford University Press
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    Publication Date: 2016-11-17
    Description: Pseudomonas putida KT2440 is a saprophytic and generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene cloning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/ loxP site-specific recombination. PCR-based Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Here we report an improved scarless gene deletion strategy based on recombineering and intron-encoded homing endonuclease I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for a 54.8 kb deletion, and the efficiency of RecA-mediated double-strand break repair, which was boosted by Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study, and biotechnological and environmental applications of P. putida KT2440.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 6
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    Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA (2016)
    Oxford University Press
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    Publication Date: 2016-11-09
    Description: Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at –20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 7
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    The endolysin from the Enterococcus faecalis bacteriophage VD13 and conditions stimulating its lytic activity (2016)
    Oxford University Press
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    Publication Date: 2016-10-12
    Description: Bacteriophages produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage endolysin. VD13 endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 endolysin. The predicted size of VD13 endolysin is ~27 kDa as verified by SDS-PAGE. The VD13 endolysin lyses Enterococcus faecalis strains, but not E. faecium or other non-enterococci. VD13 endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 endolysin, in ammonium acetate (C 2 H 3 O 2 NH 4 ) calcium chloride (CaCl 2 ) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 min, whereas activity is lost upon heating to 42°C, in pH 7 buffer.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 8
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    A simple technique for suppressor detection in Escherichia coli (2016)
    Oxford University Press
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    Publication Date: 2016-10-12
    Description: To study the viability of a gyrA S83 stop mutation found in an Escherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in the bla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codon gyrA and allow viability. The method was applied to 22 unique clinical E. coli isolates, and all were found to have low-level suppressor activity.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 9
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    Epigenetics knocks on synthetic biology's door (2016)
    Oxford University Press
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    Publication Date: 2016-09-08
    Description: Epigenetics is the study of heritable changes in gene expression without concomitant changes in DNA sequence. Due to its relevance in development, differentiation and human health, epigenetics has recently become an emerging area of science with regard to eukaryotic organisms and has shown enormous potential in synthetic biology. However, significant examples of epigenetic regulation in bacterial synthetic biology have not yet been reported. In the current study, we present the first model of such an epigenetic circuit. We termed the circuit the alternator circuit because parental cells carrying this circuit and their progeny alternate between distinct and heritable cellular fates without undergoing changes in genome sequence. Furthermore, we demonstrated that the alternator circuit exhibits hysteresis because its output depends not only on its present state but also on its previous states.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 10
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    Cultivation of an immobilized (hyper)thermophilic marine microbial community in a bioreactor (2016)
    Oxford University Press
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    Publication Date: 2016-09-08
    Description: Cultivation in a bioreactor of immobilized deep-sea hydrothermal microbial community was tested in order to assess the stability and reactivity of this new system. A community composed of eight hydrothermal strains was entrapped in a polymer matrix that was used to inoculate a continuous culture in a gas-lift bioreactor. The continuous culture was performed for 41 days at successively 60°C, 55°C, 60°C, 85°C and 60°C, at pH 6.5, in anaerobic condition and constant dilution rate. Oxic stress and pH variations were tested at the beginning of the incubation. Despite these detrimental conditions, three strains including two strict anaerobes were maintained in the bioreactor. High cell concentrations (3 x 10 8 cells mL –1 ) and high ATP contents were measured in both liquid fractions and beads. Cloning-sequencing and qPCR revealed that Bacillus sp. dominated at the early stage, and was later replaced by Thermotoga maritima and Thermococcus sp. Acetate, formate and propionate concentrations varied simultaneously in the liquid fractions. These results demonstrate that these immobilized cells were reactive to culture conditions. They were protected inside the beads during the stress period and released in the liquid fraction when conditions were more favorable. This confirms the advantage of immobilization that highlights the resilience capacity of certain hydrothermal microorganisms after a stress period.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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