实验基础

Conditional mutants are important tools particularly in the analysis of essential genes. In this chapter, a method is described that allows for a rapid design-based generation of temperature-sensitive alleles of many Saccharomyces cerevisiae genes. The method employs a temperat ...

One essential step for the molecular dissection of gene function is gene inactivation. In the yeast Saccharomyces cerevisiae, elaborate tools for gene disruption are available. Gene disruption cassettes carrying completely heterologous marker genes flanked by short DNA segme ...

The synthetic lethal screen is a method of isolating novel mutants whose survival is dependent on a gene of interest. Combining the colony-color assay with a synthetic lethal screen offers a means to visually detect a mutant that depends on a plasmid for survival. Screening for synthetic lethals ...

Proper folding, and consequently exit from the endoplasmic reticulum (ER) and secretion of heterologous exocytic proteins in yeast can be rescued by fusing the proteins to certain yeast-derived polypeptides. Biologically active mammalian glycoproteins can be produced in Sacch ...

Biochemistry is an important experimental tool in the study of protein functions. Biochemical studies frequently involve overexpression of a cloned gene and purification of the recombinant protein. The yeast Saccharomyces cerevisiae provides an effective system for express ...

The concept of telomeres as being the end-part of eukaryotic chromosomes was first described by H. J. Muller and B. McClintock (1,2). Their pioneering work opened the path for multiple new researches and assays on a thrilling subject, with implications for various domains such as aging, replicat ...

Study of gene expression can be facilitated by using a reporter gene assay. Instead of directly measuring the level of target gene mRNA, one can clone the promoter region of the gene of interest in front of a reporter gene and measure the reporter gene expression as a reflection of the expression of the gene of ...

Chromatin structure and nucleosome positioning play a crucial role in gene expression regulation. Nucleosome positioning is often inferred by the protection of underlying DNA to nucleases. Because nucleases are excluded by plasma membranes, chromatin mapping requires isola ...

Classic strain development that combines random mutagenesis and selection has a long history of success in generation of biocatalysts with industrially designed traits. However, the genetic loci contributing to the phenotypic strain changes are difficult to identify prior to gen ...

In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to suppo ...

Discovery of promoter elements with previously unknown regulated responses is important for metabolic engineering. For example, promoters responsive to the end product can be useful to regulate expression with increasing levels of product. In addition, such promoters can be used as s ...

A method for in vivo evolution of metabolic pathways in bacteria is described. This method is a powerful tool for synthetic biology type of metabolic design and can lead to the creation of new metabolic pathways or the improvement of existing metabolic enzymes. The proposed strategy also permits ...

MIRAGE is a unique in vivo genome editing technique that exploits the inherent instability of inverted repeats (palindromes) in the Saccharomyces cerevisiae chromosome. As a technique able to quickly create deletions as well as precise point mutations, it is valuable in applications t ...

A noncarotenogenic microbe E. coli was engineered for high production of carotenoids. To increase the isoprenoid flux, the chromosomal native promoters of the rate-controlling steps (dxs, idi and ispDispF) in the isoprenoid pathway were replaced with a strong bacteriophage T5 promot ...

Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. We have observed that when random mutations are not ...

There is increasing interest in utilization of engineered microorganisms for the production of renewable chemicals and next-generation biofuels. However, imbalances between the cofactor consumption of the engineered production pathway and the reducing equivalents pro ...

Cellulose is an easily renewable and highly occurring resource. To take advantage of this great potential, there is a constant need of new cellulose degrading enzymes. In industrial applications enzymes have to function under extreme conditions like high temperature, very acidic or bas ...

Phage infections in bacterial bioprocesses constitute one of the most devastating threats to the productivity of the biotechnology facilities. There are several factors, which can decide if an infection would occur, and if it would turn into an outbreak and heavy contamination of the prod ...

Escherichia coli is the most commonly used microorganism for production of recombinant proteins for different applications. Acetate accumulation during aerobic growth on glucose has significant negative impact on recombinant protein production in Escherichia coli. Vari ...

A cornerstone of Synthetic Biology is the engineering of gene regulatory networks. Construction of such biological circuits has been used not only to elucidate the dynamics of gene expression but also for designing whole-cell biosensors that translate environmental signals into qu ...