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Biochemical Characterization of Laboratory Mutants of Extended-Spectrum ß-Lactamase TEM-60. Bibiana Caporale, 2004.Three mutants of the extended-spectrum ß-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis . The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background . Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp . paratuberculosis DNA in Artificially Contaminated Milk. Jim O'Mahony, 2004.Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp . paratuberculosis DNA in artificially contaminated milk . Initially, the PCR parameters (including primer and probe levels, assay volume, Mg2+ concentration, and annealing temperature) were optimized . Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 106 to 3 x 101 copies) . The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility . A range of DNA isolation strategies was developed for isolating M . avium subsp . paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns . When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml) . Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M . avium subsp . paratuberculosis in spiked milk from heavily and mildly contaminated samples . Gene Replacement in Mycobacteria by Using Incompatible Plasmids. Carey A. Pashley, 2003.A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis . This delivery system relies on the use of a pair of replicating plasmids, which are incompatible . Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells . Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids . For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene . Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied . Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection . pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M . smegmatis and Mycobacterium bovis BCG . The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids . To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes . Using this system, we demonstrated that M . smegmatis pyrF mutants could be isolated at high frequency . This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis .
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