Table 1 Bacteria and plasmids used in the selleck kinase inhibitor study Strain or plasmid Description Source Escherichia coli 1830 pro¯ met¯ Kan r Nm r, containing transposon Tn5 on the suicidal plasmid pBJ4JI [44] DH5α supE44ΔlacU169(Φ80lacZΔM15) hsdR17recA1 gyrA96thi-1relA1 [39] BL21(DE3) hsdS gal(λcIts857 ind1 Sam7 nin5 lac UV5-T7 gene 1) [45] Pectobacterium carotovorum subsp. carotovorum
3F-3 Pcc, wild-type Laboratory stock F-rif-18 3F3, Rifr, wild-type This study TF1-1 F-rif-18, fliC::Tn5, Rifr, Kanr This study TF 1-2 F-rif-18, CarocinS2::Tn5, Rifr, Kanr This study SP33 Pcc, wild-type Laboratory stock Plasmid pMCL210 p15A, Cmlr, Low copy number [46] pGEM T-Easy Ampr; lacZ cloning vector Promega pET32a Ampr; expression vector with the N-terminal His-tag Novagen pET30b Kanr; expression vector with the C-terminal His-tag Novagen pMS2KI 5.7-kb
BamHI DNA fragment harboring carocin S2 gene from 3F3 genome, cloned into pMCL210 This study pEN2K* caroS2K subcloned into pET32a This study pES2KI Derived from pEN2K; deleted series of Tag element in front of expressed caroS2K This study pEH2KI* Derived from pES2KI; adding (His)6-Tag adjacent to caroS2I This study pGS2I caroS2I and its putative promoter from pMS2KI, subcloned into pGEM T-easy This study pECS2I* caroS2I subcloned into pET30b, but the expressed fusion CaroS2I has no activity This study pES2I Derived form pECS2I, the (His)6-Tag element was deleted This study Kanr: Kanamycin; Cmlr: Chloramphenicol; Rifr: Rifampicin; Ampr: Ampicillin. *: See Additional file 1, Figure S5. GF120918 manufacturer Bacterial conjugation Overnight cultures of Pcc (recipient) and E. coli (donor) were mixed and spread onto 0.22-μm membrane filters placed on LB agar media and incubated overnight at 28°C [23]. The Tariquidar order progeny after conjugation were appropriately diluted and cultivated Arachidonate 15-lipoxygenase on Modified Drigalski’s medium (with ampicillin and kanamycin [100 μg ml-1]) overnight at 28°C. All isolates were placed on IFO-802 medium and tested for bacteriocins. Bacteriocin was assayed using the double-layer method, and Pcc SP33 was used as indicator strain [35]. The cells were incubated for 12
hours to form colonies, exposed to ultraviolet irradiation, incubated again for 12 hours, treated with chloroform to kill the cells, and then covered with soft agar containing indicator cells. The bacteriocin production was indicated by a zone of inhibition of indicator-cell (SP33) growth around the colony. Genetic-engineering technique The procedures of plasmid preparation, genomic DNA isolation, and DNA manipulation were performed as described by Sambrook et al. [36]. Oligonucleotide DNA primers were synthesized by MD Bio Inc. (Taipei, Taiwan). The PCR was amplified with Go-Taq DNA polymerase (Promega, USA). The thermal asymmetric interlaced PCR (TAIL-PCR) was performed as previously described [37]. Plasmids were introduced into Pcc strains using electroporation (1.25 kV/cm, 200 Ω, 25 μF) [38].