TABLE 1

List of bacterial strains, plasmids and primers used

Strain, plasmid, or primerRelevant genotype, description, or sequence (5′ to 3′)aReference and/or source
Escherichia coli strains
    DH5αF λ ϕ80dlacZΔM15 endA1 recA1 hsdR17 (rK mK+) supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF)U16920
    XL10 GoldΔ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA lac, Hte, Tetr, CamrStratagene
    XL1 red competent cellsendA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac mutD5 mutS mutT Tn10, TetrStratagene
Actinomycetes
    Streptomyces lavendulae ATCC 13664Penicillin V acylase producerATCC; 7
    Streptomyces lividans 1326 (NRRL B-16148)TsrsARS culture collection (NRRL)
    Rhodococcus sp. strain T104KACC 21099KnsKorean Agricultural Culture Collection; 18
Plasmids
    pGEM-T Easy VectorLineal, T at the ends, Apr, f1, orilacZ, T3p, T7p, 3 kbPromega
    pEM4Shuttle vector for E. coli/Streptomyces, Apr, Tsrr, PermE*, pUCori, pWHM4ori, 7.9 kb16
    pNV19Shuttle vector for E. coli/Rhodococcus, Knr, pAL5000ori, lacZ, CoE1ori, 4.4 kb17
    pEPA1FpEM4 derivative containing 2,445-bp XbaI/EcoRI pva gene from S. lavendulae ATCC 13664, E. coli RBS, ATG start codon and signal peptide sequenceThis work
    pNPVA1FpNV19 derivative containing 2,249-bp HindIII/EcoRI fragment from pEPA1F including pva gene from S. lavendulae ATCC 13664, E. coli RBS, ATG start codon, signal peptide sequence, and strong constitutive PermE* promoter from Saccharopolyspora erythraeaThis work
Primers
    STREPTOP25′-CCGGAATTCAC(C/G)GAGGC(C/G)GGCATCCC(G/C)CA-3′This work
    STREPTOD25′-CCGGAATTC(C/G)GGGCCGTA(C/G)CG(C/G)GTCCACCACTG-3′This work
    STREPTOG15′-CCGGAATTCCG(C/G)GGCCT(C/G)CT(C/G)CT(C/G)GGCAACCC(C/G)CA-3′This work
    STREPTOG35′-CCGGAATTCGCC(C/G)CGGAA(C/G)GC(C/G)AC(C/G)GCGTT-3′This work
    NsacII5′-CCGGAATTCCCGCGGTGGAGACCGCCGCGT-3This work
    CsacII5′-CCGGAATTCCCGCGGGCACCGCCGCCGCC-3′This work
    FcβD25′-AGCCGGGGCGCGCTGCTCTT-3′This work
    FcβI25′-AGGCCGCCGACACGT(C/T)(G/A)AC-3′This work
    PVA-15′-TCTAGAGGGTATTAATAATGACCTTCCGTAACCGCCTCAGACTG-3′This work
    PVA-F5′-CCGGAATTCCCTACCGCCGCTCGTGCACCCG-3′This work
  • a Engineered endonuclease sites on the oligonucleotides are underlined, RBS are in italics, and start and stop codons are in boldface.