Small-molecule screening campaigns of model bacteria have been conducted extensively in biotechnology and pharmaceutical companies to search for novel compounds with antibacterial activity. Recently, there has been increasing interest in running such high-throughput s ...
To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activ ...
Zebrafish represents a versatile model organism with many molecular, morphological, and physiological similarities to mammals. Importantly, zebrafish are readily susceptible to perturbations by small molecules, including numerous pharmaceuticals in clinical use. Gi ...
The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the ...
Fluorescence-based assays are widely used in high-throughput screening due to their high sensitivity, diverse selection of fluorophores, ease of operation, and various readout modes. As a result, fluorescence-based assays have been applied to monitor a broad range of activities in li ...
The small plant Arabidopsis thaliana has been an indispensable tool for plant biologists working in fields that utilize cell biology, molecular biology, and genetics; these topics are almost universal in plant biology studies, ranging from genomics to ecology. In this chapter, we present a ...
Overexpression of heterologous proteins in the yeast Saccharomyces cerevisiae often inhibits its growth, while inhibitors of the overexpressed proteins can restore growth. These simple observations form the basis of a technically easy, inexpensive, scalable, and widely appl ...
Reporter gene assays are versatile and sensitive methods of assaying numerous targets in high-throughput drug-screening programs. A variety of reporter genes allow users a choice of signal that can be tailored to the required sensitivity, the available detection apparatus, the cell ...
Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligonucleotide recombination is one type of reco ...
Reconstructing Evolutionary Adaptive Paths (REAP) is one of several methods to improve enzyme �functionality. This approach incorporates computational and theoretical aspects of protein engineering to create a focused library of protein variety with a high degree of function ...
The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enh ...
In the past decade, in vitro evolution techniques have been used to improve the performance or alter the activity of a number of different enzymes and have generated enzymes de novo. In this review, we provide an overview of the available in vitro methods, their application, and some general conside ...
TALENs, fusion proteins of DNA binding domains of TAL (transcription activator-like) effectors and the DNA cleavage domains of endonuclease FokI, have emerged as genetic tools for targeted gene modification, holding great potential for basic and applied research, even for gene thera ...
A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to �understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, s ...
There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes ...
Isothermal titration calorimetry (ITC) has emerged as a powerful tool for determining the thermodynamic properties of chemical or physical equilibria such as protein–protein, ligand–receptor, and protein–DNA binding interactions. The utility of ITC for determining kinetic ...
We describe here a genetic selection system that directly links protein stability to antibiotic resistance, allowing one to directly select for mutations that stabilize proteins in vivo. Our technique is based on a tripartite fusion in which the protein to be stabilized is inserted into the m ...
A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-we ...
Low protein solubility is a problem in many areas of protein science. Although chemical methods have been developed to solubilize proteins these are not always effective and add to the cost of producing the protein. One way of overcoming these difficulties is to evolve the protein to be more solubl ...
A new method for protein sequence diversification is based on generating random codon mutations to an encoding DNA. This allows for the scanning of user-defined amino acid changes to any protein of interest, and is an alternative to traditional directed evolution strategies. This chapter d ...
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