Joint Canada-Israel Health Research Program / Programme conjoint canado-israélien de recherche en santé

Permanent URI for this collection

https://hdl.handle.net/10625/57460

Browse

Recent Submissions

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    High-efficiency transformation and expression of genomic libraries in yeast
    (2023-09-21) Loock, Mira; Antunes, Luiza Berenguer; Heslop, Rhiannon T.; De Lauri, Antonio Alfonso; Lira, Andressa Brito; Cestari, Igor; Levine, Stuart
  • Thumbnail Image
    Item
    Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis
    (2021-01) Levy, Shani; Brekhman, Vera; Bakhman, Anna; Malik, Assaf; Sebé-Pedrós, Arnau
  • Thumbnail Image
    Item
    Genome-wide libraries for protozoan pathogen drug target screening using yeast surface display
    (American Chemical Society, 2023-04-21) Heslop, Rhiannon ; Gao, Mengjin; Lira, Andressa Brito; Sternlieb, Tamara; Loock, Mira; Sanghi, Sahil Rao; Cestari, Igor
  • Thumbnail Image
    Item
    BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes
    (2021-04-14) Wright, Shane C.; Lukasheva, Viktoriya; Le Gouill, Christian; Kobayashi, Hiroyuki; Breton, Billy; Kobilka, Brian K.
  • Thumbnail Image
    Item
    High-efficiency transformation and expression of genomic libraries in yeast
    (MDPI, 2023-09-21) Loock, Mira; Antunes, Luiza Berenguer; Heslop, Rhiannon T; De Lauri, Antonio Alfonso; Lira, Andressa Cestari; Brito, Igor; Levine, Stuart
    Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 du/ µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 du/ µg plasmid DNA. The protocol generates sufficient transformants for 10-100 x coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method' s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for down stream screening purposes while limiting the amount of the genomic library required.
  • Thumbnail Image
    Item
    Low-dose penicillin in early life induces long-term changes in murine gut microbiota, brain cytokines and behavior
    (Springer Nature - Nature Communications, 2017-04) Leclercq, Sophie; Mian, Firoz M.; Stanisz, Andrew M.; Bindels, Laure B.; Cambier, Emmanuel; Ben-Amram, Hila; Koren, Omry; Forsythe, Paul; Bienenstock, John
    There is increasing concern about potential long-term effects of antibiotics on children’s health. Epidemiological studies have revealed that early-life antibiotic exposure can increase the risk of developing immune and metabolic diseases, and rodent studies have shown that administration of high doses of antibiotics has long-term effects on brain neurochemistry and behaviour. Here we investigate whether low-dose penicillin in late pregnancy and early postnatal life induces long-term effects in the offspring of mice. We find that penicillin has lasting effects in both sexes on gut microbiota, increases cytokine expression in frontal cortex, modifies blood–brain barrier integrity and alters behaviour. The antibiotic-treated mice exhibit impaired anxiety-like and social behaviours, and display aggression. Concurrent supplementation with Lactobacillus rhamnosus JB-1 prevents some of these alterations. These results warrant further studies on the potential role of early-life antibiotic use in the development of neuropsychiatric disorders, and the possible attenuation of these by beneficial bacteria.