We describe the construction and validation of five mini-Tn7 vectors for analysis of post-transcriptional gene expression in Pseudomonas. Four vectors allow construction of translational fusions to β-galactosidase (lacZ), while the fifth is designed for functional analysis of noncoding RNA genes. Translational fusions can be constructed without a functional promoter in the vector or from an inducible promoter of either P(tac) or P(dctA). We show that promoterless fusions have value for determining levels of translation, whereas fusions to inducible promoters have utility in the analysis of mRNA-binding factors.
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http://dx.doi.org/10.1016/j.mimet.2014.10.015 | DOI Listing |
Appl Environ Microbiol
October 2024
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal Tn site without the need for selection pressure.
View Article and Find Full Text PDFACS Synth Biol
September 2024
Department of Plant Pathology, Entomology, and Microbiology, Iowa State University, Ames, Iowa 50011, United States.
KT2440 (formerly ) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/, LacI/, and AraC/) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome.
View Article and Find Full Text PDFJ Vis Exp
January 2024
Department of Microbiology, University of Manitoba;
Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A.
View Article and Find Full Text PDFMethods Protoc
March 2019
Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi-do 13488, Korea.
Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases).
View Article and Find Full Text PDFAppl Environ Microbiol
June 2019
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of In this study, mini-Tn vectors with zeocin and apramycin selection markers were created by cloning the and genes, respectively, enabling either inducible gene expression (pUC18T-mini-TnT-Zeo-LAC and pUC18T-mini-TnT-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-TnT-Zeo and pUC18T-mini-TnT-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn vectors described above.
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