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Cold Spring Harbor Protocols Aug 2017In this introduction we discuss some basic genetic tools and techniques that are used with the fission yeast Genes commonly used for selection or as reporters are...
In this introduction we discuss some basic genetic tools and techniques that are used with the fission yeast Genes commonly used for selection or as reporters are discussed, with an emphasis on genes that permit counterselection, intragenic complementation, or colony-color assays. is most stable as a haploid organism. We describe its mating-type system, how to perform genetic crosses and methods for selecting and propagating diploids. We discuss the relative merits of tetrad dissection and random spore preparation in strain construction and genetic analyses. Finally, we present several types of mutant screens, with an evaluation of their respective strengths and limitations in the light of emerging technologies such as next-generation sequencing.
Topics: Crosses, Genetic; Genes, Mating Type, Fungal; Genetic Complementation Test; Genetics, Microbial; Molecular Biology; Schizosaccharomyces; Selection, Genetic
PubMed: 28765303
DOI: 10.1101/pdb.top079772 -
Cold Spring Harbor Protocols Jul 2017Here we provide methods for setting up standard crosses with strains. All strain genotypes and pedigrees should be recorded in a laboratory strain book. Matings between...
Here we provide methods for setting up standard crosses with strains. All strain genotypes and pedigrees should be recorded in a laboratory strain book. Matings between two haploid strains of interest are induced on solid medium poor in nitrogen. Usually, sporulation agar (SPA) plates are preferred, but for difficult matings it is advisable to try several mating media in parallel because one medium might allow for more efficient mating. Protoplast fusion can be used to produce zygotes from sterile mutants that fail to mate.
Topics: Crosses, Genetic; Culture Media; Genetics, Microbial; Schizosaccharomyces
PubMed: 28679701
DOI: 10.1101/pdb.prot091694 -
Croatian Medical Journal Aug 2005The field of microbial forensics was formalized because of the need for attribution in events where a bioweapon has been used. Microbial forensics has its origins in... (Review)
Review
The field of microbial forensics was formalized because of the need for attribution in events where a bioweapon has been used. Microbial forensics has its origins in traditional forensics, microbiology, and epidemiology. Microbial forensics can be defined as a scientific discipline dedicated to analyzing evidence for attribution purposes from a bioterrorism act, biocrime, hoax, or inadvertent microorganism/toxin release. This is a very challenging task, since there are myriad microorganisms that can pose a threat, and analytical methods need to be used reliably. The Scientific Working Group on Microbial Genetics and Forensics (SWGMGF) has addressed some quality assurance and control issues, and particularly validation criteria (focusing on preliminary validation) due to the dynamic nature of evolving investigations. Unique identification of a microorganism may never be possible. Yet, qualitative and/or quantitative assessments of the evidence can be made. One approach to provide direction on gaps in the microbial forensics effort is to perform an end-to-end retrospective analysis of past cases. As an example, the case of a gastroenterologist who was accused of second degree attempted murder of his paramour using HIV as the weapon was reviewed. The scientific evaluation involves epidemiology, molecular biology, phylogenetics, and legal deliberations.
Topics: Bioterrorism; Crime; Forensic Medicine; Genetics, Microbial; HIV; HIV Seropositivity; Humans; United States
PubMed: 16100753
DOI: No ID Found -
Applied Microbiology and Biotechnology Mar 2012Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of... (Review)
Review
Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.
Topics: Gene Expression; Genetic Engineering; Genetic Vectors; Genetics, Microbial; Mutagenesis, Insertional; Plasmids; Recombination, Genetic; Synthetic Biology; Yeasts
PubMed: 22311648
DOI: 10.1007/s00253-012-3920-8 -
British Medical Bulletin Jan 1962
Topics: Bacteria; Drug Therapy; Genetics, Microbial; Humans
PubMed: 14474649
DOI: 10.1093/oxfordjournals.bmb.a069939 -
The Journal of Antimicrobial... Apr 2003
Review
Topics: Amidohydrolases; Anti-Infective Agents; Drug Delivery Systems; Drug Design; Enzyme Inhibitors; Genetics, Microbial; Genomics; Methionine-tRNA Ligase; Oligonucleotide Array Sequence Analysis
PubMed: 12654742
DOI: 10.1093/jac/dkg178 -
Journal of Industrial Microbiology &... Dec 2010This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the... (Review)
Review
This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.
Topics: Academies and Institutes; Czech Republic; Genetics, Microbial; History, 20th Century; Molecular Biology
PubMed: 21086098
DOI: 10.1007/s10295-010-0859-6 -
Medical Mycology Jan 2017Research on fungal pathogens with the aim to identify virulence determinants strictly relies on the generation of defined, recombinant strains, a task that is executed... (Review)
Review
Research on fungal pathogens with the aim to identify virulence determinants strictly relies on the generation of defined, recombinant strains, a task that is executed by means of a sophisticated molecular biology toolbox. Recent developments in fungal genome engineering have opened a new frontier by implementing the CRISPR-Cas9 technology, based on expression of the Cas9 endonuclease that is loaded by a single guiding RNA (sgRNA) molecule to target a defined site in the recipient genome. This novel approach has been adapted successfully to engineer fungal genomes, among them the one of the human-pathogenic mould Aspergillus fumigatus Implementation of the required components was achieved by various means that differ with respect to expression of the Cas9 enzyme and sgRNA delivery. Validation of CRISPR-Cas9-mediated mutagenesis could be executed by targeting selected candidate genes of A. fumigatus to provide a promising perspective for screening and multiplexing approaches to scrutinize the virulome of this opportunistic fungal pathogen in a comprehensive manner, such as by analyzing genetic polymorphisms or the function of gene families.
Topics: Aspergillus fumigatus; Gene Targeting; Genetics, Microbial; Mutagenesis
PubMed: 27811178
DOI: 10.1093/mmy/myw097 -
Cold Spring Harbor Protocols Aug 2017Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for cells.
Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for cells.
Topics: Ethyl Methanesulfonate; Genetics, Microbial; Mutagenesis; Mutagens; Schizosaccharomyces
PubMed: 28765296
DOI: 10.1101/pdb.prot091736 -
Antonie Van Leeuwenhoek Oct 2012This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA... (Review)
Review
This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomycete genes.
Topics: Actinobacteria; Genetic Engineering; Genetics, Microbial; Genome, Bacterial
PubMed: 22926833
DOI: 10.1007/s10482-012-9795-y