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In JoVE (3)
- Большой Вставить экологического производства геномной библиотеки
- Экспрессия рекомбинантных белков в метилотрофных дрожжей Pichia pastoris
- Экран с высоким Пропускная Biomining Целлюлаза активность метагеномных из библиотеки
Other Publications (5)
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Articles by Marcus Taupp in JoVE
JoVE General
Большой Вставить экологического производства геномной библиотеки
Department of Microbiology and Immunology, University of British Columbia - UBC
Строительство fosmid библиотека с экологическими геномной ДНК, выделенной из вертикального континуума глубины сезонно гипоксических фьорда описано. В результате клон библиотеки собирают в 384-луночных планшетах и архивируются для последующих последовательности и функционального скрининга путем применения автоматизированной системы сбора колонии.
JoVE General
Экспрессия рекомбинантных белков в метилотрофных дрожжей Pichia pastoris
Department of Microbiology and Immunology, University of British Columbia - UBC
Протокол описывает экспрессии белка использованием метилотрофных дрожжей
JoVE General
Экран с высоким Пропускная Biomining Целлюлаза активность метагеномных из библиотеки
Microbiology and Immunology, University of British Columbia - UBC
Этот протокол описывает экран высокого пропускную способность для целлюлолитических деятельности от метагеномных библиотеки выражается в кишечной палочки. Экран урегулирования, основанного и высокой степенью автоматизации, и использует одну-пот в 384 химии и планшеты с окончательным считывания как измерения абсорбции.
Other articles by Marcus Taupp on PubMed
Growth, Virulence, and Immunogenicity of Listeria Monocytogenes Aro Mutants
Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10(4)-fold for the aroA, aroB, and aroA/B mutants and >10(5)-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 x 10(6) aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 x 10(8) aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.
Opposite Enantioselectivities of Two Phenotypically and Genotypically Similar Strains of Pseudomonas Frederiksbergensis in Bacterial Whole-cell Sulfoxidation
Soil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strains that preferentially produced the S-configured sulfoxide enantiomer were isolated. Surprisingly, one bacterial strain, genotypically and phenotypically characterized as Pseudomonas frederiksbergensis, selectively gave the R enantiomer. The finding that two apparently identical organisms displayed opposite enantioselectivities is novel for non-genetically modified organisms.
Production of Natural Methyl Anthranilate by Microbial N-demethylation of N-methyl Methyl Anthranilate by the Topsoil-isolated Bacterium Bacillus Megaterium
Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of 70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No byproducts were observed. Thus, production of "natural" methyl anthranilate using B. megaterium is a significant improvement over comparable already existing procedures.
Direct Trapping of Formaldehyde Formed Via Oxidative N-demethylation of N,N-dialkylarylamines by Bacillus Megaterium Using Cysteamine Derivatization
Oxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylamines N,N-dimethylaniline and N-ethyl-N-methylaniline. Formed formaldehyde, normally difficult to analyse in biological systems because of further metabolization, was successfully trapped and converted into thiazolidine by addition of cysteamine into the incubation media. Studies using N,N-di-(trideutero-methyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di-[methyl-(13)C]-aniline and N-ethyl-N-[methyl-(13)C]-aniline were performed to confirm that the N-demethylation proceeds via formaldehyde.
The Art and Design of Functional Metagenomic Screens
This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host-vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.
