Characterizing and Improving pET Vectors for Cell-free Expression.

Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

Short communication: Typing and tracking Bacillaceae in raw milk and milk powder using pyroprinting.

Contamination of fluid and processed milk products with endospore-forming bacteria, such as Bacillaceae, affect milk quality and longevity. Contaminants come from a variety of sources, including the dairy farm environment, transportation equipment, or milk processing machinery. Tracking the origin of bacterial contamination to allow specifically targeted remediation efforts depends on a reliable strain-typing method that is reproducible, fast, easy to use, and amenable to computerized analysis. Our objective was to adapt a recently developed genotype-based Escherichia coli strain-typing method, called pyroprinting, for use in a microbial source-tracking study to follow endospore-forming bacillus bacteria from raw milk to powdered milk. A collection of endospores was isolated from both raw milk and its finished powder, and, after germination, the vegetative cells were subject to the pyroprinting protocol. Briefly, a ribosomal DNA intergenic transcribed spacer present in multiple copies in Bacillaceae genomes was amplified by the PCR. This multicopy locus generated a mixed PCR product that was subsequently subject to pyrosequencing, a quantitative real-time sequencing method. Through a series of enzymatic reactions, each nucleotide incorporation event produces a photon of light that is quantified at each nucleotide dispensation. The pattern of light peaks generated from this mixed template reaction is called a pyroprint. Isolates with pyroprints that match with a Pearson correlation of 0.99 or greater are considered to be in the same group. The pyroprint also contains some sequence data useful for presumptive species-level identification. This method identified groups with isolates from raw milk only, from powdered milk only, or from both sources. This study confirms pyroprinting as a rapid, reproducible, automatically digitized tool that can be used to distinguish bacterial strains into taxonomically relevant groups and, thus, indicate probable origins of bacterial contamination in powdered milk.

Pyroprinting: a rapid and flexible genotypic fingerprinting method for typing bacterial strains.

Bacterial strain typing is commonly employed in studies involving epidemiology, population ecology, and microbial source tracking to identify sources of fecal contamination. Methods for differentiating strains generally use either a collection of phenotypic traits or rely on some interrogation of the bacterial genotype. This report introduces pyroprinting, a novel genotypic strain typing method that is rapid, inexpensive, and discriminating compared to the most sensitive methods already in use. Pyroprinting relies on the simultaneous pyrosequencing of polymorphic multicopy loci, such as the intergenic transcribed spacer regions of rRNA operons in bacterial genomes. Data generated by sequencing combinations of variable templates are reproducible and intrinsically digitized. The theory and development of pyroprinting in Escherichia coli, including the selection of similarity thresholds to define matches between isolates, are presented. The pyroprint-based strain differentiation limits and phylogenetic relevance compared to other typing methods are also explored. Pyroprinting is unique in its simplicity and, paradoxically, in its intrinsic complexity. This new approach serves as an excellent alternative to more cumbersome or less phylogenetically relevant strain typing methods.

Uptake and transmission of Toxoplasma gondii oocysts by migratory, filter-feeding fish.

From bottlenose dolphins, to walruses, to sea otters, the parasitic protozoan Toxoplasma gondii is infecting marine mammals around the world. Whereas the terrestrial transmission pathways of T. gondii are well-described, the transmission pathway by which marine mammals are being infected is unknown. We hypothesize that migratory filter feeders, specifically northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax), are serving as biotic vectors for T. gondii within the marine environment. By filtering oocysts from seawater, these fishes could be transporting the oocysts from nearshore to pelagic environments. In this study, we experimentally exposed northern anchovies and Pacific sardines to T. gondii oocysts under laboratory conditions. Following exposure, the fishes' alimentary canals were harvested and assayed for the presence of T. gondii by PCR. Fish exposed to as few as 1197 oocysts/L seawater tested positive for T. gondii by PCR. In total, the PCR assay detected T. gondii DNA in 66% (40/61) of the exposed fishes. Oocyst infectivity was confirmed by mouse bioassay: 30% (7/23) of mice developed toxoplasmosis when fed fish exposed to 100,000 oocysts/L. This study demonstrates that both northern anchovies and Pacific sardines can filter T. gondii oocysts out of seawater under experimental conditions. Our experiments with anchovies demonstrated that the oocysts persisted in the fish for at least 8h post-exposure and our experiments with sardines demonstrated that the oocysts remained infectious inside the fish's alimentary canals.

Cloning yeast actin cDNA leads to an investigative approach for the molecular biology laboratory.

The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support.

Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes.

The endocytic pathway in yeast leads to the vacuole, but resident proteins of the late Golgi, and some endocytosed proteins such as the exocytic SNARE Snc1p, are retrieved specifically to the Golgi. Retrieval can occur from both a late pre-vacuolar compartment and early or 'post-Golgi' endosomes. We show that the endosomal SNARE Pep12p, and a mutant version that reaches the cell surface and is endocytosed, are retrieved from pre-vacuolar endosomes. As with Golgi proteins, this requires the sorting nexin Grd19p and components of the retromer coat, supporting the view that endosomal and Golgi residents both cycle continuously between the exocytic and endocytic pathways. In contrast, retrieval of Snc1p from post-Golgi endosomes requires the sorting nexin Snx4p, to which Snc1p can be cross-linked. Snx4p binds to Snx41p/ydr425w and to Snx42p/ydl113c, both of which are also required for efficient Snc1p sorting. Our findings suggest a general role for yeast sorting nexins in protein retrieval, rather than degradation, and indicate that different sorting nexins operate in different classes of endosomes.