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One-Step Inactivation of Chromosomal Genes in Escherichia coli K-12 Using PCR Products
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
Journal Article
Herding Hemingway's cats : understanding how our genes work
The language of genes has become common parlance. We know they make your eyes blue, your hair curly or your nose straight. The media tells us that our genes control the risk of cancer, heart disease, alcoholism or Alzheimer's. The cost of DNA sequencing has plummeted from billions of pounds to a few hundred, and gene-based advances in medicine hold huge promise. We've all heard of genes, but how do they actually work? Drawing on stories ranging from six toed cats and stickleback hips to Mickey Mouse mice and zombie genes - told by researchers working at the cutting edge of genetics - Kat Arney explores the mysteries in our genomes with clarity, flair and wit, creating a companion reader to the book of life itself.
Book
Correction: SRY gene isolation from teeth for forensic gender identification-An observational study
[This corrects the article DOI: 10.1371/journal.pone.0294751.].
Journal Article
Correction: The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach)
[This corrects the article DOI: 10.1371/journal.pgen.1011011.].
Journal Article
Efficient protein production by yeast requires global tuning of metabolism
The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion.
Journal Article
Correction: Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato
[This corrects the article DOI: 10.1371/journal.pone.0231961.].
Journal Article