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Validation of the Psa mutant of interest (MOI) library.

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posted on 2017-03-01, 18:44 authored by Carl H. Mesarich, Jonathan Rees-George, Paul P. Gardner, Fatemeh Ashari Ghomi, Monica L. Gerth, Mark T. Andersen, Erik H. A. Rikkerink, Peter C. Fineran, Matthew D. Templeton

(A) PCR screen to identify disruptions in the IYO_023025 gene. Pooled genomic DNA samples from the columns (lanes 1–12) and rows (lanes A–H) of MOI library plate 3 (P3) were used as templates for PCR. Two sets of amplicons that share a specific IYO_023025 disruption across a single pooled column and row sample are boxed in green and red, respectively; (B) Location of the P3-G10 and P3-D4 wells, which contain a mutant with an IYO_023025 disruption specific to the PCR amplicons boxed in green and red in (A), respectively; (C) Schematic of the IYO_023025 gene showing the location of transposon insertion sites identified in (A). Transposon insertion sites are denoted by arrows, and are color-coded to match the PCR amplicons boxed green and red in (A); (D) PCR screen to identify the IYO_023025 disruption mutant from well P3-G10. Pooled genomic DNA samples from the columns and rows of a 96-well plate (P2) containing independent colony-forming units of well P3-G10 were used as templates for PCR; (E) Location of intersecting wells in P2 (dark green) that possibly contain the IYO_023025 P3-G10 disruption mutant, as determined by the PCR amplicon profile in (D); (F) PCR screen to determine which of the intersecting wells in (E) contain the IYO_023025 P3-G10 disruption mutant. Amplicons shown in the left and right panels (separated by a black line) are derived from different regions of the same gel. Wells that contain the mutant are shown in bold in (E); (G) Colony morphology of wild-type (WT) Psa and the IYO_023025 P3-G10 disruption mutant. Bar = 2 μM, M = DNA ladder, bp = base pairs,– = H2O negative control, WT = WT Psa DNA.

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