Extended Data Fig. 1: Mtb hypomorph strain creation.

a, Hypomorph strains were constructed by introducing a DAS tag at the 3ʹ end of the gene of interest, with concomitant introduction of a 20-nucleotide barcode and an episomally encoded, regulated SspB gene to control the level of protein depletion. b, Degradation of a DAS-tagged target gene product was mediated by SspB, the expression of which was driven by an ATC-inducible TetON promoter. To allow individualized degrees of knockdown for each gene product, a series of TetON promoters with varying strengths was generated. Regulated promoter strength was quantified by fusion to a luciferase gene and measuring luminescence in the presence and absence of the ATC inducer. In the screen, strains containing the TetON-1, -2, -6, -10 and -18 promoters were used for subsequent strain construction. Independent biological replicates (n = 8) are shown as open circles; means are shown as horizontal bars; error bars denote 95% confidence intervals. c, A range of up to five different knockdown levels was attempted using five different promoters for each target gene, allowing the generation of 2,014 hypomorphs. d, Barcoded hypomorph strains were pooled and distributed into 384-well plates containing the compound library and incubated for 14 days. e, Chromosomal strain barcodes were inserted into each engineered hypomorph, thus allowing PCR amplification by an array of primers containing 5′ overhangs encoding screen ___location (well and plate) barcodes. For census enumeration of pooled strains, PCR products were combined and subjected to Illumina NGS. f, Dose response of trimethoprim, a DHFR inhibitor, against wild-type Mtb and the DHFR hypomorph, showing the hypersensitivity of the hypomorph. Growth was calculated as OD₆₀₀ normalized to untreated controls. Independent biological replicates (n = 4) are shown as open circles; means are shown as filled circles; error bars denote 95% confidence intervals.

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