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CDR1 gene deletion in C. auris . Wild-type C.auris was transformed with a cassette designed to replace CDR1. (A) Nourseothricin resistant transformants were transferred to YPD + cyclohexi(i)mide (50μg/ml). Seven of the transformants that were sensitive to cyclohexi(i)mide are shown here. Diagnostic PCR using genomic DNA extracted from wild-type (W+) and transformants tested in (A) using oligonucleotide primers (B) CDOUTF2/WCLOXR, (C) WCLOXF2/CDOUTR and (D) CdrFor/CdrRev (C). Sizes of markers (M) are indicated on the left.

Journal: bioRxiv

Article Title: Construction of a drug sensitised Candida auris strain

doi: 10.1101/2024.12.28.630619

Figure Lengend Snippet: CDR1 gene deletion in C. auris . Wild-type C.auris was transformed with a cassette designed to replace CDR1. (A) Nourseothricin resistant transformants were transferred to YPD + cyclohexi(i)mide (50μg/ml). Seven of the transformants that were sensitive to cyclohexi(i)mide are shown here. Diagnostic PCR using genomic DNA extracted from wild-type (W+) and transformants tested in (A) using oligonucleotide primers (B) CDOUTF2/WCLOXR, (C) WCLOXF2/CDOUTR and (D) CdrFor/CdrRev (C). Sizes of markers (M) are indicated on the left.

Article Snippet: Genomic DNA was extracted using the YeaStar TM Genomic DNA extraction kit (Zymo Research, USA) and used as template for diagnostic PCR to confirm locus-specific integration of the deletion cassette.

Techniques: Transformation Assay, Diagnostic Assay

MDR1 gene deletion in C. auris . (A) Wild-type C.auris was transformed with a cassette designed to replace MDR1. (A) Nourseothricin resistant transformants were transferred to YPD + cyclohexi(i)mide (50μg/ml). (B) Diagnostic PCR using genomic DNA extracted from W+ and three transformants tested in (A) using oligonucleotide primers MdrF2/MdrR3. Sizes of markers (M) are indicated on the left.

Journal: bioRxiv

Article Title: Construction of a drug sensitised Candida auris strain

doi: 10.1101/2024.12.28.630619

Figure Lengend Snippet: MDR1 gene deletion in C. auris . (A) Wild-type C.auris was transformed with a cassette designed to replace MDR1. (A) Nourseothricin resistant transformants were transferred to YPD + cyclohexi(i)mide (50μg/ml). (B) Diagnostic PCR using genomic DNA extracted from W+ and three transformants tested in (A) using oligonucleotide primers MdrF2/MdrR3. Sizes of markers (M) are indicated on the left.

Article Snippet: Genomic DNA was extracted using the YeaStar TM Genomic DNA extraction kit (Zymo Research, USA) and used as template for diagnostic PCR to confirm locus-specific integration of the deletion cassette.

Techniques: Transformation Assay, Diagnostic Assay

Removal of the NAT1 drug resistance marker gene from cdr1Δ::NAT1-Clox. (A) Cells were grown on YPD with (B) and without (A) 200 μg/mL nourseothricin at 30°C for 5 days. Wild-type (W+) C. auris and cdr1Δ::NAT1-Clox display nourseothricin sensitivity and resistance, respectively, as expected. cdr1Δ::lox P was generated by resolution of the NAT1 marker gene via Cre-mediated recombination across lox P sites. (B) Diagnostic PCR using genomic DNA extracted from W+ and cdr1Δ::lox P using oligonucleotide primers CDOUTF2/CDOUTR.

Journal: bioRxiv

Article Title: Construction of a drug sensitised Candida auris strain

doi: 10.1101/2024.12.28.630619

Figure Lengend Snippet: Removal of the NAT1 drug resistance marker gene from cdr1Δ::NAT1-Clox. (A) Cells were grown on YPD with (B) and without (A) 200 μg/mL nourseothricin at 30°C for 5 days. Wild-type (W+) C. auris and cdr1Δ::NAT1-Clox display nourseothricin sensitivity and resistance, respectively, as expected. cdr1Δ::lox P was generated by resolution of the NAT1 marker gene via Cre-mediated recombination across lox P sites. (B) Diagnostic PCR using genomic DNA extracted from W+ and cdr1Δ::lox P using oligonucleotide primers CDOUTF2/CDOUTR.

Article Snippet: Genomic DNA was extracted using the YeaStar TM Genomic DNA extraction kit (Zymo Research, USA) and used as template for diagnostic PCR to confirm locus-specific integration of the deletion cassette.

Techniques: Marker, Generated, Diagnostic Assay

Generating a cdr1Δmdr1Δ double delete. The cdr1Δ:lox P was transformed with a cassette designed to replace MDR1 . (B) Of one hundred and twenty nourseothricin resistant colonies, three were hypersensitive to 1 μg/mL cyclohexi(i)mide. Growth of these three strains compared to W+, cdr1Δ::lox P, mdr1Δ::NAT1-Clox on (A) YPD and (B) YPD+1 μg/mL cyclohexi(i)mide (C) Diagnostic PCR using genomic DNA extracted from the strains indicated in (A) using oligonucleotide primers MdrF2/MdrR3.

Journal: bioRxiv

Article Title: Construction of a drug sensitised Candida auris strain

doi: 10.1101/2024.12.28.630619

Figure Lengend Snippet: Generating a cdr1Δmdr1Δ double delete. The cdr1Δ:lox P was transformed with a cassette designed to replace MDR1 . (B) Of one hundred and twenty nourseothricin resistant colonies, three were hypersensitive to 1 μg/mL cyclohexi(i)mide. Growth of these three strains compared to W+, cdr1Δ::lox P, mdr1Δ::NAT1-Clox on (A) YPD and (B) YPD+1 μg/mL cyclohexi(i)mide (C) Diagnostic PCR using genomic DNA extracted from the strains indicated in (A) using oligonucleotide primers MdrF2/MdrR3.

Article Snippet: Genomic DNA was extracted using the YeaStar TM Genomic DNA extraction kit (Zymo Research, USA) and used as template for diagnostic PCR to confirm locus-specific integration of the deletion cassette.

Techniques: Transformation Assay, Diagnostic Assay