Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing?

The empirical and pragmatic nature of diagnostic microbiology has given rise to several different schemes to subtype E.coli, including biotyping, serotyping, and pathotyping. These schemes have proved invaluable in identifying and tracking outbreaks, and for prognostication in individual cases of infection, but they are imprecise and potentially misleading due to the malleability and continuous evolution of E. coli. Whole genome sequencing can be used to accurately determine E. coli subtypes that are based on allelic variation or differences in gene content, such as serotyping and pathotyping. Whole genome sequencing also provides information about single nucleotide polymorphisms in the core genome of E. coli, which form the basis of sequence typing, and is more reliable than other systems for tracking the evolution and spread of individual strains. A typing scheme for E. coli based on genome sequences that includes elements of both the core and accessory genomes, should reduce typing anomalies and promote understanding of how different varieties of E. coli spread and cause disease. Such a scheme could also define pathotypes more precisely than current methods.

An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source.

In this study, we describe the development of a cost effective and highly productive cell-free protein synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3mg/mL of protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this cell-free synthesis technique a promising alternative for more efficient protein production.

Economical parallel protein expression screening and scale-up in Escherichia coli.

A novel microfermentation and scale-up platform for parallel protein production in Escherichia coli is described. The vertical shaker device Vertiga, which generates low-volume high density (A(600) approximately 20) Escherichia coli cultures in 96-position deep-well plates without auxiliary oxygen supplementation, has been coupled to a new disposable shake flask design, the Ultra Yield flask, that allows for equally high cell culture densities to be obtained. The Ultra Yield flask, which accommodates up to 1 l in culture volume, has a baffled base and a more vertical wall construction compared to traditional shake flask designs. Experimental data is presented demonstrating that the Ultra Yield flask generates, on average, an equivalent amount of recombinant protein per unit cell culture density as do traditional shake flask designs but at a substantially greater amount per unit volume. The combination of Vertiga and the Ultra Yield flask provides a convenient and scalable low-cost solution to parallel protein production in Escherichia coli.