pbluescriptiiks  (Thermo Fisher)

Journal: Microbiology

Article Title: Gene ssfg_01967 ( miaB ) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672

doi: 10.1099/mic.0.000747

Figure Lengend Snippet: Strains and plasmids used in this work

Article Snippet: Bacterial strain or plasmid Description* Source or reference E. coli DH5α supE44 ΔlacU169(φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 NEB E. coli ET12567 (pUZ8002) dam -13::Tn9(Cm r ) dcm -6 hsd M; harbors conjugative plasmid pUZ8002; Cm r , Km r C.P.Smith, UMIST, UK; E. coli WM6026 lacI q rrnB3 Δ lacZ 4787 hsdR514 Δ araBAD567 Δ rhaBAD568 rph-1 att λ::pAE12 (Δ ori R6K- cat ::Frt5) Δ endA ::Frt uidA (ΔMluI):: pir att HK::pJK1006Δ ( ori R6K- cat ::Frt5 trfA ::Frt) [ 58 ] E. coli BW25113 (pKD46) (F - (φ80dΔ( lacZ )M15 recA1 endA1 gyrA96 thi1 deoR ( lacZYA-argF )U169); λ bet- exo -gam; aph (Km r ) J. Beckwith, HMS S. ghanaensis ATCC14672 Wild-type moenomycin producer ATCC S. ghanaensis B38.3 Markerless deletion of moeH5 ; produces NoA [ 21 ] S. ghanaensis HIM5 Himar1 insertion into ssfg_01967 gene of B38.3 This work Bacillus cereus ATCC19637 Moenomycin-sensitive test culture ATCC pTES Am r ; phiC31-based Streptomyces integrative vector; expression of cloned gene from ermEp * [ 59 ] pKC1132 Am r ; conjugative suicide vector for gene disruption [ 14 ] pIJ6902 Am r Th r ; phiC31-based Streptomyces integrative vector; expression of cloned gene from tipAp [ 60 ] pHYG pLitmus-derived vector containing hygromycin resistance cassette hyg ; Hy r Ap r J. Salas, Universidad de Oviedo patt-shyg hygromycin cassette flanked by B-CC and P-GG sites for IMES recombination system [ 61 ] pGUS Promoter probe vector, contains promoterless gusA ; derived from pSET152 [ 20 ] pGUSHL4aadA pGUS derivative for translational fusions [ 20 ] padpAscript pGUS derivative, transcriptional fusion of adpAp (0.5 kb KpnI-XbaI fragment) to gusA , Am r Sp r [ 8 ] padpAtransl pGUSHL4aadA derivative, adpA-gusA translational fusion, Am r Sp r [ 8 ] padpAcontr pGUSHL4aadA, promoterless adpA gh -gusA fusion, Am r Sp r [ 8 ] pmiaB pKC1132 carrying 5 kb BamHI-HindIII fragment centred on ssfg_01967, Am r This work pmiaBhyg pmiaB derivative, replacement of with hyg cassette from patt-shyg, Am r Hy r This work pBluescriptIIKS Ap r ; general purpose cloning vector; blue–white selection ThermoScientific pOOB120 pBluescriptIIKS carrying 1.8 kb adpA-adpA fragment This work pOOB121 pOOB120 with TTA codon within adpA replaced with CTC This work padpAtransl-CTC pGUSHL4aadA carrying adpAp-adpA from pOOB121 This work pOOB106 pTES carrying gene ssfg_01967 under ermEp * promoter This work pOOB106a pOOB106 with aac(3)IV marker changed to hyg This work pOOB108L pIJ6902 carrying gene ssfg_01967 under tipAp promoter This work pOOB113a pOOB108L with aac(3)IV marker changed to hyg This work Open in a separate window *Cmr, Kmr, Amr, Spr, Thr, Hyr, Apr: chloramphenicol, kanamycin, apramycin, spectinomycin, thiostrepton, hygromycin and ampicillin resistance, respectively.

Techniques: Plasmid Preparation, Expressing, Clone Assay, Disruption, Derivative Assay, Cloning, Selection, Marker