hygromycin  (Gold Biotechnology Inc)

Journal: Frontiers in Microbiology

Article Title: Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

doi: 10.3389/fmicb.2016.01951

Figure Lengend Snippet: C. guilliermondii pmr1 Δ null mutant shows increased susceptibility to cell wall perturbing agents . The C. guilliermondii strains ATCC 6260, KU141 (Parental), HMY134 ( pmr1 Δ), and HMY138 ( pmr1 Δ + PMR1 ) were incubated with different concentrations of either Calcofluor White, Congo Red, Tunicamycin, or Hygromycin B, and growth was determined after incubation for 24 h at 30°C. Growth data were normalized as percentage of those generated with the same strain without treatment. For data normalization, growth results are shown as percentage of those obtained with the same strain growing in the absence of any perturbing agent. Data are means ± SD of three independent experiments performed in duplicates. For all the agents tested, the null mutant sensitivity was significantly different to that of the control strains ( P

Article Snippet: The maximum concentrations tested for each agent were: 400 μg/mL Calcofluor White (CFW, Sigma), 200 μg/mL Congo Red (Sigma), 300 μg/mL hygromycin B (GoldBio); 50 μg/mL tunicamycin (Sigma) and 0.1% (w/v) SDS (Invitrogen).

Techniques: Mutagenesis, Incubation, Generated

Journal: The Plant journal : for cell and molecular biology

Article Title: Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779

doi: 10.1111/tpj.14314

Figure Lengend Snippet: A one-vector CRISPR system for gene disruption in N. oceanica . (A) The pNOC-CRISPR vector series includes a Hygromycin B resistance cassette (HygC, green). A bidirectional promoter (Ribi) drives the transcription of the Cas9-reporter fusion and gRNA scaffold (scaffold, blue), along with the LDSP and CS terminators (LDSP-T, CS-T) respectively. Cas9 is fused to either the GFP or Nlux (with HA tag) reporters by a 3x glycine-serine linker (GSGSGS) and contains SV40 nuclear localization signals on the N’ and C’ termini (NLS). The gRNA scaffold and the 3’ self-cleaving HDV ribozyme is integrated into the vector. The 5’ hammerhead ribozyme (HH) specific for each guide sequence (GS, orange) is fused to the gRNA scaffold (scaffold) to form a sgRNA. Ribozymes are highlighted in yellow. Unique restriction sites are shown with an upwards line and the name in italics. (B) Confocal analysis of DAPI nuclear staining, Cas9-GFP signal, and merged brightfield, DAPI and GFP signal in N. oceanica cells. Scale bar of 2 μM. (C) Immunoblotting with an α-GFP antibody detected the Cas9-GFP produced in N. oceanica transformed with pNOC-CRISPR-GFP. A N. oceanica line producing a delta-5 fatty acid desaturase (~75 kDa) fused with CFP was used as a GFP positive control (+). (D) Immunoblotting with an α-HA antibody detected the appropriately sized Cas9-Nlux-HA in N. oceanica transformed with pNOC-CRISPR. Wild-type N. oceanica was included as a negative control (WT). For (C) and (D) numbers on the left of immunoblots indicate size markers (KDa).

Article Snippet: Individual lines were transferred to 500 μl of F/2 with 100 μg/ml Hygromycin B (GoldBio) in a 96 deep well plate (Evergreen Biotech).

Techniques: Plasmid Preparation, CRISPR, Sequencing, Staining, Produced, Transformation Assay, Positive Control, Negative Control, Western Blot

Journal: The Plant journal : for cell and molecular biology

Article Title: Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779

doi: 10.1111/tpj.14314

Figure Lengend Snippet: Generation of marker-free non-transgenic mutants by episomal removal (curing). (A) Luminescence from equal number of cells of wild-type (WT), and NR-KO lines either containing the episome or cured of the episome. (B) PCR for detection of a positive control NR genomic locus and the Cas9 regions on the episome conducted on the same DNA extract obtained from WT and, NR-KO episomal and cured lines. (C) Plating of an equal number of cells of WT, NR-KO and NR-KO cured lines on NH 4 , NO 3 , and NH 4 with Hygromycin B on F/2 solid medium after 1 month of growth.

Article Snippet: Individual lines were transferred to 500 μl of F/2 with 100 μg/ml Hygromycin B (GoldBio) in a 96 deep well plate (Evergreen Biotech).

Techniques: Marker, Transgenic Assay, Polymerase Chain Reaction, Positive Control

Journal: Frontiers in Microbiology

Article Title: Role of Protein Mannosylation in the Candida tropicalis-Host Interaction

doi: 10.3389/fmicb.2019.02743

Figure Lengend Snippet: Susceptibility to cell wall perturbing agents in the Candida tropicalis pmr1 Δ and och1 Δ null mutants. Yeast cells were incubated in YPD broth supplemented with different concentrations of Congo red, Calcofluor white, or Hygromycin B, and cell growth was determined after incubation for 24 h at 30°C. For normalization, growth results are shown as a percentage of those obtained with the same strain grown in the absence of any perturbing agent. Data are means ± SD of three independent experiments performed in duplicates. Both null mutant strains showed increased susceptibility to the three cell wall perturbing agents analyzed, and were significantly different to either the WT or the reintegrant control strain ( P

Article Snippet: Calcofluor white and Congo red were from Sigma; whereas hygromycin B from GoldBio.

Techniques: Incubation, Mutagenesis