TABLE 7

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant characteristic(s)aReference or source
Strains
    E. coli BW25113lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD7816
    E. coli BL21(DE3)F ompT hsdSB (rB mB) gal dcm rne131 (DE3)Laboratory storage
    E. coli EB205Derived from BW25113; ΔyqhD::atoB ΔldhA::hbd Δ(ackA-pta)::crt ΔadhE::ter ΔfrdBC::adhE2 ΔeutE::fdhThis work
    E. coli EB215Derived from EB205; Δ(hyc-hyp)::FRT ΔfdhF::fdhThis work
    E. coli EB216Derived from EB215; Δmdh::FRTThis work
    E. coli EB216-Tn5-599#Derived from EB216; pykA gene was inserted using a Tn5 transposonThis work
    E. coli EB216-ΔpykADerived from EB216; ΔpykAThis work
    E. coli EB216-ΔpykFDerived from EB216; ΔpykFThis work
    E. coli EB216-ΔyhjXDerived from EB216; ΔyhjXThis work
    E. coli EB216-ΔpykA&pykFDerived from EB216-ΔpykA; ΔpykFThis work
    E. coli EB216-ΔpykA&yhjXDerived from EB216-ΔpykA; ΔyhjXThis work
Plasmids
    pKmTsSacBSkeleton plasmid for cloning, constructed from pK18mobsacB (ATCC) with thermosensitive ori of pKD46, kan20
    pKmTsSacB-yqhD::atoBpKmTsSacB-based vector, containing yqhDup-atoB-yqhDdown cassette, kanThis work
    pKmTsSacB-ldhA::hbdpKmTsSacB-based vector, containing ldhAup-hbd-ldhAdown cassette, kanThis work
    pKmTsSacB-(ackA-pta)::crtpKmTsSacB-based vector, containing (ackA-pta)up-crt-(ackA-pta)down cassette, kanThis work
    pKmTsSacB-adhE::terpKmTsSacB-based vector, containing adhEup-ter-adhEdown cassette, kanThis work
    pKmTsSacB-frdBC::adhE2pKmTsSacB-based vector, containing frdBCup-adhE2-frdBCdown cassette, kanThis work
    pKmTsSacB-eutE::fdhpKmTsSacB-based vector, containing eutEup-fdh-eutEdown cassette, kanThis work
    pKmTsSacB-fdhF::fdhpKmTsSacB-based vector, containing fdhFup-fdh-fdhFdown cassette, kanThis work
    pKD4bla, FRT, kan16
    pKD46bla, araC, gam-bet-exo16
    pCP20bla, flp, cat16
    pET30akanLaboratory storage
    pET30a-pykApET30a-based vector, containing His-tagged PykA gene, kanThis work
    pET30a-pykFpET30a-based vector, containing His-tagged PykF gene, kanThis work
  • a bla, ampicillin resistance gene; kan, kanamycin resistance gene; gam-bet-exo, Red recombinase genes; flp, flipase (Flp recombinase) gene; cat, chloramphenicol resistance gene; FRT, flipase recognition target.