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Table 1 Plasmids used in this study

From: Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria

Plasmids provided by others or reported earlier:
Name Relevant features References
pABB19 oriV MB1, ApR, derivative of cloning vector pUC19 with added transcription termination sequence T pro /T lyz from P1 [49]
pABB705 pKRP10 derivative with inactivated NcoI and PvuII sites in CmR cassette [49]
pALA136 oriV MB1, oriV P1, CmR , dual replicon [9]
pBGS18 oriV MB1, KmR, cloning vector [56]
pBAD24 oriV MB1, ApR, araC, araBADp, expression vector [38]
pCM132 oriV ColE1, oriV RK2, oriT RK2, traJ’, trfA, KmR, promoter-less lacZ, dual replicon [52]
pGBT30 oriV MB1, ApR, lacI q , tacp expression vector [34]
pGEM-T-Easy oriV MB1, ApR, cloning vector Promega
pJSB1.24 pBGS18 tra RA3 korC RA3 (RA3 coordinates 9437- 33857; 3093-3705) [49]
pKLB3 pGBT30 tacp-parA parB P.a [29]
pKRP10 oriV MB1, ApR, CmR [50]
pMPB9.90 pBAD42 araBADp-trfA RK2 Przyluski M.
pPT01 oriV pSC101, KmR, promoter-less xylE [10]
pRK415 oriV RK2, TcR, oriT RK2, stable vector [16]
pYC16A pALA136 with RA3 stabilization region [41]
RA3 IncU, CmR, SmR, SuR [40]
RK2 IncP-1α, KmR, ApR, TcR Thomas C.M.
Plasmids constructed during this work:
  Description, relevant features
pABB18.1 pPT01 klcAp -xylE, PCR fragment amplified with primers #1 and #2 on RA3 template inserted as SphI-BamHI fragment
pABB18.2 pABB18.1 cleaved with HpaI and NcoI, filled in and self-ligated to remove 561 bp upstream of xylE
pABB18.3 pABB19 with klcAp-xylE inserted as SmaI-PscI PCR fragment amplified with primers #3 and #4 on pABB18.2 template
pABB18.4 pBGS18 with korB RK2 inserted as EcoRI-SalI PCR fragment amplified with primers #5 and #6 on RK2 template
pABB18.5 pGEM-T-Easy with lacI q gene PCR-amplified primers #11 and #12 on pGBT30 template
pABB25 pAKB20.1 with 527-bp NcoI fragment removed
pABB25.1 pABB25 with cat gene (CmR) on BamHI fragment from pABB705 replacing BclI fragment from TcR cassette
pABB26 pABB25.1 with XhoI restriction site introduced downstream of trbA (PCR directed mutagenesis with primers #20 and #21)
pABB27 pABB26 with ApaI restriction site downstream of korA gene (PCR directed mutagenesis with primers #22 and #23)
pABB28 pABB27 with klcp-xylE cassette, SmaI-NcoI fragment from pABB18.3
pABB29 pABB28 with korB RK2 gene, ApaI-SalI fragment from pABB18.4 inserted between ApaI and XhoI sites
pABB32 pABB29 with MCS flanked with lacO operators inserted between BssHII and NcoI sites, unstable vector
pABB33 pABB32 with lacI q -tacp-parA-parB P.a. , DraI-SalI fragment of pKLB3 inserted between SnaBI and XhoI sites
pABB34 pABB33 with parS P.a., annealed oligonucleotides #9 and #10 inserted into BglII restriction site
pABB35 pABB32 with lacI q, NruI-PvuI fragment of pABB18.5 cloned between SmaI and PvuI sites, unstable vector
pAKB17.9 pABB32 with RA3 active partition cassette (korA-incC-korB-orf11-parS), EcoRV-BamHI fragment from pYC16A inserted between Ecl136II and BglII sites
pAKB17.10 pABB35 with RA3 active partition cassette (korA-incC-korB-orf11-parS), EcoRV-BamHI fragment from pYC16A inserted between Ecl136II and BglII sites
pAKB20.1 pRK415 TcR with trfAp-1 introduced by PCR mutagenesis with primers #18 and #19 and spontaneous deletion of 1974-bp fragment encompassing MCS, traJ and oriT
pESB3.6 pUC18 with synthetic RA3 partition cassette (korA-incC-korB-orf11-parS), cloned between EcoRI and SalI sites (RA3 coordinates 5940-9800)
pESB30 pABB35 with oriT RK2 , 218-bp fragment PCR-amplified on RK2 template using primers #13 and #14, cleaved with PscI and cloned into NcoI site, unstable, RK2 mobilizable vector
pESB31 pABB35 with oriT RA3, 166-bp fragment PCR-amplified on RA3 template using primers #15 and #16, cleaved with PscI and cloned into NcoI site, unstable, RA3 mobilizable vector
pESB32 pESB30 with klcAp-lacZ, BglII-NcoI fragment from derivative of pCM132 inserted between BamHI and PscI sites, unstable, RK2 mobilizable vector
pESB34 pESB32 with synthetic RA3 partition cassette (korA-incC-korB-orf11-parS), EcoRI-SalI fragment from pESB3.6 cloned between EcoRI and XhoI sites
pJSB1.28 pJSB1.24 Ppu618, 618-nt PCR-amplified fragment of P. putida chromosome, coordinates 58074-58691
  1. Vectors for cloning and analysis of stabilization cassettes are in bold