微生物学技术

This chapter describes the RNA sequencing (RNA-Seq) protocol, whereby RNA from yeast cells is prepared for sequencing on an Illumina Genome Analyzer. The protocol can easily be altered to use RNA from a different organism. This chapter covers RNA extraction, cDNA synthesis, cDNA fragmenta ...

Cellular networks and processes can be mathematically described and analyzed in various ways. Here, the case example of a MAP kinase (MAPK) cascade is used to detail steps in the formulation of a system of ordinary differential equations governing the temporal behavior of a signal transduct ...

One of the major objectives of systems biology is the development of mathematical models for the quantitative description of complex biological systems, such as living cells. Biological data and software tools for the design, analysis, and simulation of models are two basic ingredients f ...

The rapidly increasing availability of DNA sequence data from modern high-throughput experimental techniques has created the need for computational algorithms to aid in motif discovery in genomic DNA. Such algorithms are typically used to find a statistical representation of the n ...

The yeast Saccharomyces cerevisiae is the model organism in which protein interactions have been most extensively analyzed. The vast majority of these interactions have been characterized by a variety of sophisticated high-throughput techniques probing different aspects of p ...

One of the major aims of the nascent field of evolutionary systems biology is to test evolutionary hypotheses that are not only realistic from a population genetic point of view but also detailed in terms of molecular biology mechanisms. By providing a mapping between genotype and phenotype for ...

The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has be ...

We describe a human cell display strategy to isolate high-affinity single-chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient p ...

Phage display has the capacity to rapidly isolate recombinant antibodies against protein targets and other molecules of significant size. However, there is no obvious lower limit to the power of the selection methods: this chapter describes how the techniques of phage display can be adapt ...

The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the nov ...

This chapter describes the construction and screening of a library of single-chain variable fragments (svFv) derived from patients with autoimmune disease. The methods cover the isolation of mononuclear cells from peripheral blood, preparation of RNA, and recovery of immunoglob ...

A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a r ...

A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries gene ...

Organisms have the ability to counteract environmental perturbations and keep certain components within a cell homeostatically regulated. Closely related to homeostasis is the behavior of perfect adaptation where an organism responds to a step-wise perturbation by regulati ...

In this chapter, stochasticity in gene expression is investigated using -expansion technique. Two theoretical models are considered here, one concern the stochastic fluctuations in a single-gene network with negative feedback regulation, and the other the additivity of noise pr ...

To understand the behavior of genes, it is important to explore how the patterns of gene expression change over a period of time because biologically related gene groups can share the same change patterns. In this study, the problem of finding similar change patterns is induced to clustering with t ...

Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosm ...

Gene functional studies consist of phenotyping cells with altered gene expression. Improving the precision of current gene expression control techniques would enable more detailed studies of gene function. Here, we provide protocols for building synthetic gene constructs for t ...

This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces ...

Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic construc ...