实验基础

Methods for manipulating and fermenting microorganisms in multi-well plates offer unlimited possibilities for high-throughput parallel experimentation. Furthermore, bar-coded data tracking and downstream processing with modern liquid handling equipment reduce ...

Growth media and incubation conditions have a very strong influence of secondary metabolite production. There is no consensus on which media are the optimal for metabolite production, but a series of useful and effective media and incubation conditions have been listed here. Chemically ...

The accelerating pace of microbial genomics is sparking a renaissance in the field of natural products research. Researchers can now get a preview of the organism’s secondary metabolome by analyzing its genomic sequence. Combined with other -omics data, this approach may provide a cost-e ...

Fungi produce an astonishing variety of secondary metabolites, some of which belong to the most toxic compounds in the living world. However, the benefits of secondary metabolites for fungi are often obscure. Here we describe a method that will aid in the analysis of fungal gene expression upon in ...

The hollow fiber assay (HFA) is a drug discovery tool to aid investigators in the prioritization of lead compounds identified by in vitro testing for further development in animal models of disease. In the HFA, cells are cultured in hollow fibers containing pores of a diameter (500 kDa) large enough f ...

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and ...

Recent studies conducted in our laboratory demonstrate that Aspergillus parasiticus synthesizes and stores aflatoxin in transport vesicles and endosomes. Proteomics data suggest that enzymes involved in the synthesis of other secondary metabolites as well as enzymes invol ...

Understanding in vivo protein–protein interactions is critical to dissect precise functions of the regulatory proteins of fungal secondary metabolites. As many fungi differentially produce a diverse array of secondary metabolites during their lifecycle, it is important to un ...

A crucial part of optimization of metabolically engineered organisms is producing balanced levels of pathway proteins. Typically, protein levels are monitored by Western blot analysis; however, application to multiple enzyme pathways can be difficult without unique antibodi ...

Chromatin immunoprecipitation (ChIP) is used to map the interaction between proteins and DNA at a specific genomic locus in the living cell. The protein–DNA complexes are stabilized already in vivo by reversible crosslinking and the DNA is sheared by sonication or enzymatic digestion in ...

For many fungi the number of known secondary metabolites is surprisingly small compared to the �astonishingly large number of terpene cyclase, polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS) secondary metabolite gene clusters found in their genomes. Corr ...

Fungal molecular biology has benefited from the enormous advances in understanding protein–protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extra ...

With the completion of genomes of various Aspergillus species, large-scale genome-wide expression studies can be carried out. Genomics, however, is more powerful and efficient when combined with genetics. A multi-copy-based gain-of-function screen is a complementary method to lo ...

Expression of multiple genes involved in a particular metabolic pathway in a heterologous host facilitates the study of fungal secondary metabolite biosynthesis and production of useful compounds. Two plasmids with different selection markers, argB and the pyrithiamine resis ...

Plasmid vector construction is an essential step for molecular microbiology yet often a time-consuming process. Manipulation of the fungal genome to express genes to activate secondary metabolite production often requires creation of plasmid constructs in a reiterative fashi ...

Targeted gene deletions and promoter replacements are proving to be a valuable tool for awakening and analyzing silent secondary metabolism gene clusters in Aspergillus nidulans and, as molecular genetic methods for manipulating the genomes of other fungi are developed, they will li ...

Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium abundant in soils and water. Frequent and early colonization of the human intestinal flora is common and often asymptomatic. Antimicrobials given commonly disrupt the intestinal microflora and t ...

Never before has there been a more timely opportunity to investigate the molecular genetics of Clostridium difficile. Over the last few years the perception of C. difficile has changed from an obscure, and often under-researched, bacterium to one of major clinical importance, at least in indu ...

Clostridium difficile infection (CDI) occurs as a disease with a spectrum of severity ranging from mild, self-limiting diarrhoea to a severe colitis, pseudomembraneous colitis or toxic megacolon. The disease arises as a major complication of antibiotic therapy and is most commonly ac ...

Clostridium difficile is a pathogen on the move, as evidenced by the rapid transcontinental spread of the so-called hypervirulent 027 strains, followed by the emergence of further PCR ribotypes such as 017, 078 and 106. This provides a rare opportunity to study the evolution of virulence in acti ...