Highly Multiplexed, Semiautomated Nextera Next-Generation Sequencing (NGS) Library Preparation.

High-throughput, inexpensive DNA sequencing is an essential component of large-scale DNA assembly operations. Using traditional and acoustic liquid-handling robotics, Illumina's Nextera Tagmentation reactions can be miniaturized and paired with custom PCR index primers to produce highly multiplexed NGS libraries for pooled sequencing. This chapter describes a high-throughput protocol that enables the simultaneous sequencing of thousands of DNA constructs in a single sequencing run at a dramatically reduced cost compared to bench-top methods.

High-Throughput DNA Assembly Using Yeast Homologous Recombination.

Yeast homologous recombination is a reliable, low-cost, and efficient method for DNA assembly. Using homology regions as short as 24 base pairs, constructs of up to 12 unique parts can be assembled into a diverse range of vectors. The simplicity and robustness of this protocol make it amenable to laboratory automation and high-throughput operations.

Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli.

Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in engineered microbial systems since its formation is unavoidable in terpene biosynthesis. In this work, we develop an experimental platform to study IPP toxicity in isoprenol-producing Escherichia coli. We first characterize the physiological response to IPP accumulation, demonstrating that elevated IPP levels are linked to growth inhibition, reduced cell viability, and plasmid instability.

The Experiment Data Depot: A Web-Based Software Tool for Biological Experimental Data Storage, Sharing, and Visualization.

Although recent advances in synthetic biology allow us to produce biological designs more efficiently than ever, our ability to predict the end result of these designs is still nascent. Predictive models require large amounts of high-quality data to be parametrized and tested, which are not generally available. Here, we present the Experiment Data Depot (EDD), an online tool designed as a repository of experimental data and metadata.

Characterizing Strain Variation in Engineered E. coli Using a Multi-Omics-Based Workflow.

Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield.

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.

Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E.

Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.

Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels.

Isoprenoid drugs, biofuels, and chemicals--artemisinin, farnesene, and beyond.

Isoprenoids have been identified and used as natural pharmaceuticals, fragrances, solvents, and, more recently, advanced biofuels. Although isoprenoids are most commonly found in plants, researchers have successfully engineered both the eukaryotic and prokaryotic isoprenoid biosynthetic pathways to produce these valuable chemicals in microorganisms at high yields. The microbial synthesis of the precursor to artemisinin--an important antimalarial drug produced from the sweet wormwood Artemisia annua--serves as perhaps the most successful example of this approach.

Correlation analysis of targeted proteins and metabolites to assess and engineer microbial isopentenol production.

The ability to rapidly assess and optimize heterologous pathway function is critical for effective metabolic engineering. Here, we develop a systematic approach to pathway analysis based on correlations between targeted proteins and metabolites and apply it to the microbial production of isopentenol, a promising biofuel. Starting with a seven-gene pathway, we performed a correlation analysis to reduce pathway complexity and identified two pathway proteins as the primary determinants of efficient isopentenol production.

Less is more: reduced catechol production permits Pseudomonas putida F1 to grow on styrene.

Pseudomonas putida F1 is unable to grow on styrene due to the accumulation of 3-vinylcatechol, a toxic metabolite that is produced through the toluene degradation (tod) pathway and causes catechol-2,3-dioxygenase (C23O) inactivation. In this study, we characterized a spontaneous F1 mutant, designated SF1, which acquired the ability to grow on styrene and did not accumulate 3-vinylcatechol. Whereas adaptation to new aromatic substrates has typically been shown to involve increased C23O activity or the acquisition of resistance to C23O inactivation, SF1 retained wild-type C23O activity.

Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme.

Rationale: The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations.

Bacterial strategies for growth on aromatic compounds.

Although the biodegradation of aromatic compounds has been studied for over 40 years, there is still much to learn about the strategies bacteria employ for growth on novel substrates. Elucidation of these strategies is crucial for predicting the environmental fate of aromatic pollutants and will provide a framework for the development of engineered bacteria and degradation pathways. In this chapter, we provide an overview of studies that have advanced our knowledge of bacterial adaptation to aromatic compounds.

Growth of Pseudomonas putida F1 on styrene requires increased catechol-2,3-dioxygenase activity, not a new hydrolase.

Pseudomonas putida F1 cannot grow on styrene despite being able to degrade it through the toluene degradation (tod) pathway. Previous work had suggested that this was because TodF, the meta-fission product (MFP) hydrolase, was unable to metabolize the styrene MFP 2-hydroxy-6-vinylhexa-2,4-dienoate. Here we demonstrate via kinetic and growth analyses that the substrate specificity of TodF is not the limiting factor preventing F1 from growing on styrene.

Microbial O-methylation of the flame retardant tetrabromobisphenol-A.

We demonstrated the O-methylation of tetrabromobisphenol-A (TBBPA) [4,4'-isopropylidenebis (2,6-dibromophenol)] to its mono- and dimethyl ether derivatives by microorganisms present in different sediments. A most probable number assay of a marsh sediment suggested that up to 10% of the total aerobic heterotrophs may be capable of O-methylation. Although TBBPA dimethyl ether is not produced in industry, it has been detected in terrestrial and aquatic sediments.