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

Bartosik, Aneta A.; Glabski, Krzysztof; Kulinska, Anna; Lewicka, Ewa; Godziszewska, Jolanta; Markowska, Aleksandra; Jagura-Burdzy, Grazyna

doi:10.1186/s12866-016-0674-y

BMC Microbiology

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, Ap^R, 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 Cm^R cassette	[49]
pALA136	oriV _MB1, oriV _P1, Cm^R _, dual replicon	[9]
pBGS18	oriV _MB1, Km^R, cloning vector	[56]
pBAD24	oriV _MB1, Ap^R, araC, araBADp, expression vector	[38]
pCM132	oriV _ColE1, oriV _RK2, oriT _RK2, traJ’, trfA, Km^R, promoter-less lacZ, dual replicon	[52]
pGBT30	oriV _MB1, Ap^R, lacI ^q , tacp expression vector	[34]
pGEM-T-Easy	oriV _MB1, Ap^R, 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, Ap^R, Cm^R	[50]
pMPB9.90	pBAD42 araBADp-trfA _RK2	Przyluski M.
pPT01	oriV _pSC101, Km^R, promoter-less xylE	[10]
pRK415	oriV _RK2, Tc^R, oriT _RK2, stable vector	[16]
pYC16A	pALA136 with RA3 stabilization region	[41]
RA3	IncU, Cm^R, Sm^R, Su^R	[40]
RK2	IncP-1α, Km^R, Ap^R, Tc^R	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 (Cm^R) on BamHI fragment from pABB705 replacing BclI fragment from Tc^R 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 Tc^R 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

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ISSN: 1471-2180

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