Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale.

The Resistome: A Comprehensive Database of Escherichia coli Resistance Phenotypes.

The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of information. To address this issue, we introduce the Resistome database: a literature-curated collection of Escherichia coli genotypes-phenotypes containing over 5,000 mutants that resist hundreds of compounds and environmental conditions.

Quantifying complexity in metabolic engineering using the LASER database.

We previously introduced the LASER database (Learning Assisted Strain EngineeRing, https://bitbucket.org/jdwinkler/laser_release) (Winkler et al. 2015) to serve as a platform for understanding past and present metabolic engineering practices.

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice.

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation.

A Web Interface for Codon Compression.

Saturation mutagenesis is widely used in protein engineering and other experiments. A common practice is to utilize the single degenerate codon NNK. However, this approach suffers from amino acid bias and the presence of a stop codon and of the wild type amino acid.

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli.

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli.

Complex systems in metabolic engineering.

Metabolic engineers manipulate intricate biological networks to build efficient biological machines. The inherent complexity of this task, derived from the extensive and often unknown interconnectivity between and within these networks, often prevents researchers from achieving desired performance. Other fields have developed methods to tackle the issue of complexity for their unique subset of engineering problems, but to date, there has not been extensive and comprehensive examination of how metabolic engineers use existing tools to ameliorate this effect on their own research projects.

The emergence of commodity-scale genetic manipulation.

Since the 1970s technological advancements in the fields of synthetic biology and metabolic engineering have led to a dramatic reduction in both time and cost required for generating genomic mutations in a variety of organisms. The union of genomic editing machinery, DNA inkjet printers, and bioinformatics algorithms allows engineers to design a library of thousands of unique oligos as well as build and test these designs on a ∼2 months time-scale and at a cost of roughly ∼0.3 cents per base pair.

The LASER database: Formalizing design rules for metabolic engineering.

The ability of metabolic engineers to conceptualize, implement, and evaluate strain designs has dramatically increased in the last decade. Unlike other engineering fields, no centralized, open-access, and easily searched repository exists for cataloging these designs and the lessons learned from their construction and evaluation. To address this issue, we have developed a repository for metabolic engineering strain designs, known as LASER (Learning Assisted Strain EngineeRing, laser.