falcon round bottom high clarity polypropylene tube  (Fisher Scientific)

Journal: BMC Biology

Article Title: Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

doi: 10.1186/s12915-022-01481-2

Figure Lengend Snippet: Workflow for the construction of an ordered collection of B. theta VPI-5482 transposon mutants. Single cells from a random pool of >300,000 mutants were sorted into 302 96-well plates using FACS to create a progenitor collection. After using barcode sequencing to locate strains within the progenitor collection, representative mutants for individual genes were re-arrayed to create a condensed collection of 34 96-well plates covering >2500 genes. To demonstrate the screening potential of this collection, growth and cell shape phenotypes of each strain were characterized during growth in rich medium (BHIS)

Article Snippet: Immediately before being transported to the FACS machine, log-phase cultures of the transposon library were diluted to an OD 600 of 0.01–0.05 in pre-warmed BHIS with 10 mM cysteine in a FACS tube (Falcon® high-clarity polypropylene round bottom test tubes, Corning, Cat. #352063) and mixed thoroughly in the anaerobic chamber.

Techniques: Sequencing

Journal: BMC Biology

Article Title: Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

doi: 10.1186/s12915-022-01481-2

Figure Lengend Snippet: Morphological screening reveals filamentation phenotypes of the BT0870 ( spt ) and BT2397 ( tnr3 ) mutants. A Wild-type cells become shorter and rounder in stationary phase. Phase-contrast images of log-phase cells (OD 600 ~0.4) and stationary-phase cells (OD 600 ~1.1). Scale bar: 10 μm. B (Left) BT2397 ( tnr3 ) mutant cells had higher average length and width than wild-type cells. (Right) BT0870 ( spt ) mutant cells exhibited higher variation in cell length than wild type. All strains except these two outliers are shown in gray, and wild-type replicates are shown in black . n >1500 cells per strain. C Morphological phenotypes were identified from screening. (Left) A subpopulation of BT2397 ( tnr3 ) mutant cells were longer and subpopulations of BT0870 mutant cells were longer or shorter relative to wild-type cells. n >1500 cells per strain. (Right) Representative phase-contrast images of each mutant. Scale bar: 10 μm. D Morphological phenotypes were retained after single-cell isolation. BT2397 ( tnr3 ) mutant cells were no longer elongated after passaging through a colony, but the filamentous phenotype was typically maintained after inoculating individual mutant cells into liquid BHIS using FACS with a gate to select for cells larger than wild type and culturing for 48 h. One representative culture of each strain/condition is shown and the inset shows a representative image of each culture. n >400 cells per strain

Article Snippet: Immediately before being transported to the FACS machine, log-phase cultures of the transposon library were diluted to an OD 600 of 0.01–0.05 in pre-warmed BHIS with 10 mM cysteine in a FACS tube (Falcon® high-clarity polypropylene round bottom test tubes, Corning, Cat. #352063) and mixed thoroughly in the anaerobic chamber.

Techniques: Mutagenesis, Single-cell Isolation, Passaging