实验基础

The classical strain breeding method based on random mutation and selection cannot avoid introducing detrimental or unnecessary mutations into the genome. A methodology that overcomes the limitations of the classical method is “genome breeding.” In this approach, biotechno-lo ...

A bench-scale protocol for the purification of plasmid DNA (pDNA) produced in Escherichia coli is described. The method is specifically designed to prepare pDNA vectors for gene therapy and DNA vaccination applications. The method comprises alkaline lysis, concentration with iso-p ...

Streptomyces clavuligerus is used as model to illustrate the determination and quantification of antimicrobial metabolites with different biological activities produced by cultures of this strain. S. clavuligerus produces an array of compounds with different structures. ...

Actinomycetes produce many bioactive compounds with clinical, veterinary, or agricultural applications. Many of these natural products contain sugars attached to the corresponding agly-cons, which usually participate in the molecular recognition of the cellular target. T ...

Actinomycetes are among the most fascinating microorganisms. Their developmental life cycle with its morphological and physiological differentiation and the rich repertoire of secondary metabolites (about 70-80% of bioactive secondary metabolites are being produced by ...

The ascomycete Ashbya gossypii, a filamentous fungus, is a natural overproducer of vitamin B2 and is currently exploited for the industrial production of this vitamin. Classical mutagenesis and selection of mutants showing improved production capacities have been routinely app ...

Carotenoids are high-value natural products utilized by several industrial sectors. Some filamentous fungi such as Phycomyces blakesleeanus, Mucor circinelloides, and Blakeslea trispora possess the ability to synthesise carotenoids. In this chapter, procedures for comp ...

Carotenoids are chemical compounds that are in an increasing demand in the market because of their applications in the food, feed, cosmetics, and pharmaceutical industries. To date, most of the β-carotene is manufactured by chemical processes, but because it is used as feed and food additives, t ...

A pilot-plant process for the production of a recombinant sweet-tasting protein in the filamentous fungus Aspergillus awamori is described. Each step of the scale-up and downstream processes (concentration, diafiltration and ion-exchange chromatography) for the production ...

The Gram-positive bacterium Bacillus megaterium is frequently employed for the industrial production of exoenzymes including amylases, proteinases, and penicillin acylases, for instance. Here, we describe the utilization of the excellent protein secretion capacity of this ...

This chapter introduces 13C tracer techniques combined with labeling measurement by mass spectrometry (MS) for the quantification of metabolic pathway fluxes. As an example, the relative contribution of (1) de novo synthesis from glucose and (2) synthesis from building blocks conta ...

A strain improvement procedure is outlined for the isolation of mutant strains from the bacterium Pseudomonas sp. ATCC 31461 that are capable of enhanced production of the polysaccharide gellan compared to its parent strain. With gellan having a number of industrial applications, the is ...

In this chapter are described the applications of the Morganella morganii phoC gene, encoding a molecular class A nonspecific phosphatase, as a reporter both in prokaryotic (Escherichia coli) and in eukaryotic (Saccharomyces cerevisiae) systems. The activity of PhoC can be detected by ...

Gene expression in microbes can be detected with an oligonucleotide-based DNA macroar-ray. The DNA macroarray is composed of short (22-24-mer) oligonucleotide probes immobilized onto a nylon membrane via polyinosine tails. An indirect high-density labeling method was used to effe ...

Microbial strains overproducing commercially important metabolites are routinely obtained through mutagenesis and random screening and/or selection. Advances in recombinant DNA technology have made it possible for engineering improved microbial strains by specific ...

Proteome analysis is a powerful tool for the investigation of yeast in their natural habitats. Two-dimensional gel electrophoresis has been used extensively in proteomics, but reproducibili-ty is notoriously difficult to achieve with this technology. An optimized procedure al ...

7-Aminodeacetoxycephalosporanic acid (7-ADCA) and 7-aminocephalosporanic acid (7-ACA) are the starting materials for the production of all clinically important semisynthetic derivatives of cephalosporins. Whereas 7-ADCA is conventionally produced from penicill ...

The field of predictive microbiology is rapidly widening and ranges from bioreactor engineering to the modeling of foodstuff degradation and contamination. In this chapter, we point out how important the choice of experimental conditions is. We also briefly describe some tools (meth ...

Microbiological processes and the products thereof are patentable. In this connection, &quote;products&quote; means the metabolic products of the microorganisms, the microorganisms themselves, and ingredients of the microorganisms, particularly genetic material. In ...

Patent protection can be granted for an invention that is new, of industrial value, and reworkable. To assure reworkability of an invention where biological material is involved, a sample should be deposited with an independent, recognized patent depositary. This chapter outlines in det ...