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A-B. Transcriptional repression (CRISPRi) of dxr, ispH is lethal. Guides targeting genes were cloned into the kanamycin-resistant vector pJLR965 as previously described ( , ). (A) Upon induction of CRISPR interference machinery by addition of ATc, target genes are transcriptionally repressed. (B) Quantified CFU data from CRISPRi plates +/- ATc200. Data are compiled from 5 biological replicates. C. Plasmid loss frequency analysis of a Mm mutant in Dxr, the non-mevalonate (MEP) pathway of isoprenoid biosynthesis. MmΔ dxr +p dxr , WT Mm +p dxr and WT Mm +pEmpty (empty vector) were passaged every 48hr in fresh <t>7H9</t> media without antibiotics. At each passage, cultures were serially diluted and plated on both plain and hygromycin-containing agar. Loss % < 0 indicates more CFU on hyg. N=3 biological replicates.
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A-B. Transcriptional repression (CRISPRi) of dxr, ispH is lethal. Guides targeting genes were cloned into the kanamycin-resistant vector pJLR965 as previously described ( , ). (A) Upon induction of CRISPR interference machinery by addition of ATc, target genes are transcriptionally repressed. (B) Quantified CFU data from CRISPRi plates +/- ATc200. Data are compiled from 5 biological replicates. C. Plasmid loss frequency analysis of a Mm mutant in Dxr, the non-mevalonate (MEP) pathway of isoprenoid biosynthesis. MmΔ dxr +p dxr , WT Mm +p dxr and WT Mm +pEmpty (empty vector) were passaged every 48hr in fresh <t>7H9</t> media without antibiotics. At each passage, cultures were serially diluted and plated on both plain and hygromycin-containing agar. Loss % < 0 indicates more CFU on hyg. N=3 biological replicates.
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A-B. Transcriptional repression (CRISPRi) of dxr, ispH is lethal. Guides targeting genes were cloned into the kanamycin-resistant vector pJLR965 as previously described ( , ). (A) Upon induction of CRISPR interference machinery by addition of ATc, target genes are transcriptionally repressed. (B) Quantified CFU data from CRISPRi plates +/- ATc200. Data are compiled from 5 biological replicates. C. Plasmid loss frequency analysis of a Mm mutant in Dxr, the non-mevalonate (MEP) pathway of isoprenoid biosynthesis. MmΔ dxr +p dxr , WT Mm +p dxr and WT Mm +pEmpty (empty vector) were passaged every 48hr in fresh 7H9 media without antibiotics. At each passage, cultures were serially diluted and plated on both plain and hygromycin-containing agar. Loss % < 0 indicates more CFU on hyg. N=3 biological replicates.

Journal: bioRxiv

Article Title: The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

doi: 10.1101/2025.07.11.664281

Figure Lengend Snippet: A-B. Transcriptional repression (CRISPRi) of dxr, ispH is lethal. Guides targeting genes were cloned into the kanamycin-resistant vector pJLR965 as previously described ( , ). (A) Upon induction of CRISPR interference machinery by addition of ATc, target genes are transcriptionally repressed. (B) Quantified CFU data from CRISPRi plates +/- ATc200. Data are compiled from 5 biological replicates. C. Plasmid loss frequency analysis of a Mm mutant in Dxr, the non-mevalonate (MEP) pathway of isoprenoid biosynthesis. MmΔ dxr +p dxr , WT Mm +p dxr and WT Mm +pEmpty (empty vector) were passaged every 48hr in fresh 7H9 media without antibiotics. At each passage, cultures were serially diluted and plated on both plain and hygromycin-containing agar. Loss % < 0 indicates more CFU on hyg. N=3 biological replicates.

Article Snippet: Mycobacterium marinum M (ATCC BAA-535) was routinely grown at 30°C in Middlebrook 7H9 liquid medium or 7H10 agar (BD Difco) supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase) and 0.2% Tween80.

Techniques: Clone Assay, Plasmid Preparation, CRISPR, Mutagenesis