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Spurred by recent innovations in genome sequencing, the reconstruction of genome-scale models has increased in recent years. Genome-scale models are now available for a wide range of organisms, and models have been successfully applied to a number of research topics including metabol ...

With a high demand for increasingly diverse chemicals, as well as sustainable synthesis for many existing chemicals, the chemical industry is increasingly looking to biosynthesis. The majority of biosynthesis examples of useful chemicals are either native metabolites made by an or ...

In this chapter, we describe the steps needed to create a kinetic model of a metabolic pathway using kinetic data from both experimental measurements and literature review. Our methodology is presented by using the example of serine biosynthesis in E. coli.

Introduction of a heterologous metabolic pathway into a platform microorganism for applications in metabolic engineering and synthetic biology is often technically straightforward. However, the major challenge is to balance the flux in the pathway to obtain high yield and produc ...

Complex phenotypes, such as tolerance to growth inhibitors, are difficult to rationally engineer into industrial model organisms due our poor understanding of their underlying molecular mechanisms. Adaptive evolution circumvents this issue by exploiting the linkage betwe ...

The integration of transcriptional regulatory and metabolic networks is a crucial step in the process of predicting metabolic behaviors that emerge from either genetic or environmental changes. Here, we present a guide to PROM (probabilistic regulation of metabolism), an automated ...

Recent advances in homologous recombination in Escherichia coli have enabled improved genome engineering by multiplex recombineering. In this chapter, we present trackable multiplex recombineering (TRMR), a method for gene-trait mapping which creates simulated knockdo ...

Next generation sequencing (NGS) has revolutionized the way by which we engineer metabolism by radically altering the path to genome-wide inquiries. This is due to the fact that NGS approaches offer several powerful advantages over traditional methods that include the ability to fully s ...

Directed laboratory evolution is a common technique to obtain an evolved bacteria strain with a desired phenotype. This technique is especially useful as a supplement to rational engineering for complex phenotypes such as increased biocatalyst tolerance to toxic compounds. Howev ...

Chemotaxis is the phenomenon in which cells direct their movements in the presence of certain chemicals (chemoattractants or chemorepellents). Leukocyte recruitment (via chemotaxis) is an important component of the inflammatory response, both in physiological host defense a ...

This chapter describes a method for the analysis of human hepatocarcinoma cells (Hep G2 cells) for reduced and oxidized glutathione, following treatment with nanoparticle formulations. Glutathione is a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) present intracellu ...

This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparti ...

This chapter describes a protocol for testing nanoparticle formulations for reactive oxygen species generation in male Sprague-Dawley (SD) primary hepatocytes. The protocol utilizes the fluorescent redox active probe, dichlorofluorescein diacetate (DCFH-DA). Primary ...

This chapter describes a method for monitoring nanoparticle treated human hepatocarcinoma cells (Hep G2) for apoptosis. The protocol utilizes a fluorescent method to determine the degree of caspase-3 activation.

This chapter describes method for evaluation of nanomaterial cytotoxicity by examining effects on porcine kidney (LLC-PK1) and human cancerous liver cells (Hep G2). Several studies indicate that many nanoparticles are cleared from the body through the kidney or liver, making these or ...

Sensitive and fast analytical techniques are needed to determine the concentration of nanoparticles in biological samples (e.g., blood and tissues) for biodistribution and toxicity studies. This chapter describes a method for the use of capillary zone electrophoresis (CZE) and mic ...

This chapter provides a protocol for quantitative analysis of gold in solution as well as gold uptake by macrophages. A 96-well fluorescence assay was developed to be able to determine gold concentrations for a given gold nanoparticle as well as quantify the degree of gold nanoparticle uptake by ...

This chapter describes a procedure for quantitative determination of microbial contamination of a nanoparticle formulation. The protocol includes tests for yeast, mold, and bacteria using Millipore sampler devices. This approach is primarily intended to avoid contamination ...

Bacterial endotoxin or lipopolysaccharide (LPS) is a membrane component of all Gram-negative bacteria. The administration of products contaminated with bacterial endotoxin can cause fever, shock, and even death. Accordingly, the FDA sets limits on the number of endotoxin units (EU) t ...

Energy Dispersive X-ray (EDX) microanalysis is a technique used for identification of the elemental composition of a specimen. The detection of nanoparticles in tissue is a common problem of biodistribution and toxicity studies. High-resolution transmission electron microsc ...