The Experts below are selected from a list of 156 Experts worldwide ranked by ideXlab platform

Reinhold Brückner - One of the best experts on this subject based on the ideXlab platform.

  • a new Integrative reporter Plasmid for streptococcus pneumoniae
    Fems Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid β-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia coliβ-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous β-galactosidase gene bgaA, thereby abolishing background β-galactosidase activity. The new system was tested by measuring β-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

  • A new Integrative reporter Plasmid for Streptococcus pneumoniae
    FEMS Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid beta-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia colibeta-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous beta-galactosidase gene bgaA, thereby abolishing background beta-galactosidase activity. The new system was tested by measuring beta-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

F C Minion - One of the best experts on this subject based on the ideXlab platform.

  • transformation of mycoplasma gallisepticum with tn916 tn4001 and Integrative Plasmid vectors
    Journal of Bacteriology, 1994
    Co-Authors: Paul A Kapke, F C Minion
    Abstract:

    Mycoplasma gallisepticum causes respiratory disease in avian species, but little is known about its mechanism(s) of pathogenesis. These studies were undertaken in order to develop genetic systems for analysis of potential virulence factors. M. gallisepticum was transformed with Plasmids containing one of the gram-positive transposons Tn916 or Tn4001, which inserted randomly into the mycoplasmal chromosome. Plasmids containing cloned chromosomal DNA were also constructed and tested for integration into regions of DNA homology derived either from chromosomal fragments or from the gentamicin resistance marker from Tn4001. These studies demonstrate that M. gallisepticum is amenable to transformation with both transposons and Integrative vectors. Images

  • genetic exchange of transposon and Integrative Plasmid markers in mycoplasma pulmonis
    Journal of Bacteriology, 1993
    Co-Authors: G G Mahairas, F C Minion
    Abstract:

    Abstract Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants. Transposons Tn916 and Tn4001, and a series of Integrative Plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level. Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU. Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated Plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination. Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin. Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media. The ability to exchange chromosomal markers was limited to selected strains of M. pulmonis; mating did not occur with Acholeplasma laidlawii or M. gallisepticum. Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings. The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.

Alexander Halfmann - One of the best experts on this subject based on the ideXlab platform.

  • a new Integrative reporter Plasmid for streptococcus pneumoniae
    Fems Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid β-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia coliβ-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous β-galactosidase gene bgaA, thereby abolishing background β-galactosidase activity. The new system was tested by measuring β-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

  • A new Integrative reporter Plasmid for Streptococcus pneumoniae
    FEMS Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid beta-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia colibeta-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous beta-galactosidase gene bgaA, thereby abolishing background beta-galactosidase activity. The new system was tested by measuring beta-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

Regine Hakenbeck - One of the best experts on this subject based on the ideXlab platform.

  • a new Integrative reporter Plasmid for streptococcus pneumoniae
    Fems Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid β-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia coliβ-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous β-galactosidase gene bgaA, thereby abolishing background β-galactosidase activity. The new system was tested by measuring β-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

  • A new Integrative reporter Plasmid for Streptococcus pneumoniae
    FEMS Microbiology Letters, 2007
    Co-Authors: Alexander Halfmann, Regine Hakenbeck, Reinhold Brückner
    Abstract:

    A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid beta-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia colibeta-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous beta-galactosidase gene bgaA, thereby abolishing background beta-galactosidase activity. The new system was tested by measuring beta-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new Integrative Plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

Sergey V. Mashko - One of the best experts on this subject based on the ideXlab platform.

  • Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review
    Applied Microbiology and Biotechnology, 2011
    Co-Authors: Valerii Z. Akhverdyan, Irina L. Tokmakova, Yurgis A. V. Yomantas, Nataliya V. Stoynova, Sergey V. Mashko
    Abstract:

    The advantages of phage Mu transposition-based systems for the chromosomal editing of Plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis / trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an Integrative Plasmid with a mini-Mu and an easily eliminated helper Plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans -acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells.

  • Phage Mu-driven two-Plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome.
    Applied Microbiology and Biotechnology, 2008
    Co-Authors: Elena Georgievna Abalakina, Irina L. Tokmakova, Natalya V. Gorshkova, Valerii Z. Akhverdyan, Sergey V. Mashko, Yurgis A. V. Yomantas
    Abstract:

    A phage Mu-driven two-Plasmid system for DNA integration in Escherichia coli genome has been adjusted for Methylophilus methylotrophus. Constructed helper Plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors MuA and MuB. Integrative Plasmids that are only replicated in E. coli could be mobilized to M. methylotrophus and contained mini-Mu unit with a short terminus of Mu DNA, Mu-attL/R. Mini-Mu unit was integrated in the M. methylotrophus genome via mobilization of the Integrative Plasmid to the cells carrying the helper in conditions of thermo-induced expression of MuA and MuB. In this system, mini-Mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the M. methylotrophus chromosome in the presence of helper Plasmid. A kan-gene flanked by FRT sites was inserted in one of the mini-Mu units, and it could be readily excised by yeast FLP recombinase that is encoded by the designed Plasmid. The multiple Mu-driven gene insertion was carried out by integration of the Bacillus amyloliquefaciens α-amylase gene followed by curing the KmR marker before integration of the second mini-Mu unit with Pseudomonas putida xylE gene encoding catechol 2,3-dioxygenase (C23O).