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. 2016 Jan 26;291(13):6732–6747. doi: 10.1074/jbc.M115.707810

FIGURE 6.

FIGURE 6.

Construction of Calkro_0402 knock-in C. bescii strain RKCB103. A, Calkro_0402 knock-in vector pJMC009 homologous recombination with the chromosome of parent C. bescii strain JWCB018. pJMC009 was constructed using the pDCW121 (65) vector backbone, which contains the pSC101 origin, repA, and apramycin resistance marker for maintenance in E. coli as well as C. bescii wild type pyrF under the control of the Athe_2105 promoter for selection of uracil prototrophy and 5-fluoroorotic acid counterselection. Cross-over regions 1 kb in length 5′ and 3′ to the ΔCbeI locus (ΔAthe_2438) in strain JWCB018 were used to target Calkro_0402 for insertion via homologous recombination to that locus. The Calkro_0402 insertion construct included Calkro_0402 and an additional 81 bp downstream of the gene to include its predicted transcriptional terminator element amplified from C. kronotskyensis genomic DNA. High, constitutive expression of Calkro_0402 was driven by the 200-bp promoter element upstream of the C. bescii main SLP gene (Athe_2303). B, the Calkro_0402 construct is inserted in the chromosome of RKCB103 after first crossover vector integration and second crossover 5-fluoroorotic acid counterselection to remove the vector backbone containing pyrF. C, using primers outside of the 5′ and 3′ crossover regions (Table 1, ΔAthe_2438 locus F and R), the ΔCbeI locus was amplified and sequenced to verify the Calkro_0402 knock-in. Strain JWCB018 has the expected 2.9-kb product, whereas strain RKCB103 has the expected 8.2-kb product with the 5.3-kb insertion of the SLP promoter and Calkro_0402 construct. A New England Biolabs 1-kb ladder was run alongside these PCR products. D–F, anti-Calkro_0402 immunofluorescence microscopy of C. bescii strain JWCB018, C. kronotskyensis, and C. bescii strain RKCB103 grown on birchwood xylan. D, C. bescii strain JWCB018, the genetic parent strain, which does not contain Calkro_0402, shows minimal labeling with the anti-Calkro_0402 antibodies. C. kronotskyensis (E) and Calkro_0402 knock-in C. bescii strain RKCB103 (F) are both extensively labeled with antibody. E and F, Calkro_0402 is shown localized on the cell surface in both C. kronotskyensis and C. bescii strain RKCB103 and appears primarily at the poles of the cells. All cells are labeled with chicken anti-Calkro_0402 total IgY primary antibody and DyLight 488-conjugated goat anti-chicken secondary antibody (green) with DAPI as a counterstain (blue). Scale bars, 2 μm.