Table 1.
Plasmids used in this study
| 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 Tpro/Tlyz 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 | |
Vectors for cloning and analysis of stabilization cassettes are in bold