use-of-in-vivo-assembly-for-high-efficiency-plasmid-construction

Use of <em>In Vivo</em> Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

University of Saskatchewan

 is a ubiquitous opportunistic pathogen of the family Enterobacteriaceae.  is a member of the ESKAPEE pathogens ( spp., and ), a group of bacteria that cause nosocomial infections and are able to resist killing by commonly relied upon antimicrobial agents. The acquisition of antimicrobial resistance (AMR) genes is increasing among community and clinical isolates of  making  a rising threat to human health. In addition to the increase in AMR,  is also thought to disseminate AMR genes to other bacterial species. In this review, the known mechanisms of  AMR will be described and the current state of AMR  within Canada will be discussed, including the impact of the coronavirus disease-2019 pandemic, current perspectives, and outlook for the future.

Antimicrobial resistance in : an overview of common mechanisms and a current Canadian perspective.

Plasmids are molecular genetic tools used for trans-complementation and gene expression in bacteria. Challenges faced by researchers include limited repertoire of antibiotic resistance of plasmids, issues related to plasmid compatibility and restricted or incompatible multiple cloning sites when needing to change plasmid copy number to tune production of their protein of interest. In this study, a series of plasmids were generated with compatible multiple cloning sites and homologous DNA regions to allow for modular cloning for rapid exchange of antibiotic resistance and plasmid origin. Plasmids generated in this series have options for high, mid, and low plasmid copy number, and have either an integrated FLAG epitope in the multiple cloning site or possess an uninterrupted multiple cloning site with the option of using the common LacZ-based blue/white screening method. Low copy plasmids also have one of five antibiotic selection markers. To demonstrate functionality of these plasmids, a representative FLAG tagged protein and mCherry were cloned into the low copy plasmids and expressed in various bacteria belonging to the Enterobacteriaceae family. In conclusion, by creating a new plasmid series, we have expanded the toolkit of available molecular biology tools for bacterial work.

Generation of a plasmid series for rapid sub-cloning and use in various Enterobacteriaceae.

Responsible conduct of research and ethics are key components of microbiology and are carefully considered at all steps from project conception, design, data analysis to publication, and post-publication use of samples. Yet stand-alone courses covering these topics are not always core components of undergraduate biomedical education curricula. Here, we describe a classroom activity for undergraduate students designed to promote increased understanding of ethics and experimental design in biomedical research.

Article	Year
Antimicrobial resistance in : an overview of common mechanisms and a current Canadian perspective. Canadian journal of microbiology\| PubMed ID: 39213659	2024
Generation of a plasmid series for rapid sub-cloning and use in various Enterobacteriaceae. Journal of bioscience and bioengineering\| PubMed ID: 39244484	2024
Overheard at a conference: an in-class activity to foster classroom discussion on ethics and experimental systems in microbiology research. Journal of microbiology & biology education\| PubMed ID: 39772646	2025

Jenny-Lee Thomassin

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