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Table 1 Plasmids used in this study

From: MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

Designation Relevant features Copy number References
pBBR1MCS-1 BHR1, IncA/C, CmR Medium [36]
pBGS18 ori MB1, KmR High [41]
pET28a ori MB1, KmR, T7p, lacO, His6- tag, T7 tag Medium Novagen
pGBT30 ori MB1, ApR, lacI q, tacp expression vector High [35]
pJSB1.24 ori MB1, KmR, TraRA3-korCp-korC (RA3 coordinates 9437-33657 nt, 3093-3705 nt) High [42]
pKGB4 pUT18 with MCS modified High Głąbski K.2
pKGB5 pKNT25 with MCS modified Medium Głąbski K.2
pKNT25 ori p15, KmR , lacp - MCS - cya T25 Medium [31]
pKT25 ori p15, KmR , lacp-cya T25- MCS Medium [31]
pKT25-zip ori p15, KmR , lacp-cya T25- GCN4 leucine zipper Medium [31]
pLKB2 pKT25 with MCS modified Medium Kusiak L.2
pLKB4 pUT18C with MCS modified High Kusiak L.2
pMPB13.3 ori RA3, KmR pABB20-lacI q -tacp-nic Low Przyłuski M.2
pMPB13.4 ori RA3, KmR pABB20-lacI q -tacp-mobC-nic Low Przyłuski M.2
pPT01 ori SC101, KmR, promotorless xylE Medium [50]
RA3 BHR1, IncU, CmR , SmR , SuR Low Hayes F.3
pUC18 ori MB1, ApR High [23]
pUT18 ori ColE1, ApR , lacp- MCS-cyaT18 High [31]
pUT18C ori ColE1, ApR , lacp -cyaT18- MCS High [31]
pUT18C-zip ori ColE1, ApR , lacp -cyaT18- GCN4 leucine zipper High [31]
Plasmids constructed in this study
Designation Description
pJSB2.1 pUC18-mobC, EcoRI-SalI fragment amplified by PCR with the use of primers #1 and #2 (RA3 coordinates 9837-10455 nt)
pJSB2.2 pUC18-mobC1-129, EcoRI-SalI fragment amplified by PCR with the use of primers #1 and #2 spontaneous stop codon mutation at position 10225 nt (RA3 coordinates 9837-10455 nt)
pJSB2.9 pUC18-mobCp, 417 bp SphI-BamHI fragment amplified by PCR with the use of primers #3 and #8 (RA3 coordinates 9435-9852 nt)
pJSB2.11 pUC18 with 116 bp SphI-BamHI PCR fragment amplified by PCR with the use of primers #6 and #8 (RA3 coordinates 9736-9852 nt)
pJSB2.12 pUC18 with 116 bp SphI-BamHI PCR fragment amplified by PCR with the use of primers #5 and #8 (RA3 coordinates 9736-9852 nt), mutation in a putative oriT motif
pJSB2.13 pUC18 with 100 bp SphI-BamHI PCR fragment amplified with the use of primers #5 and #4 (RA3 coordinates 9736-9836 nt), mutation in a putative oriT motif
pJSB2.14 pUC18 with 100 bp SphI-BamHI PCR fragment amplified with the use of primers #6 and #4 (RA3 coordinates 9736-9836 nt)
pJSB2.15 PCR mutagenesis of pJSB2.9 with the use of primers #9 and #10 (mutVI)
pJSB2.16 PCR mutagenesis of pJSB2.9 with the use of primers #13 and #14 (mutVII)
pJSB2.17 PCR mutagenesis of pJSB2.9 with the use of primers #11 and #12 (mutVIII)
pJSB2.18 PCR mutagenesis of pJSB2.9 with the use of primers #15 and #16 (mutIX)
pJSB2.30 pUC18 mobC1-155, EcoRI-SalI fragment amplified by PCR with the use of primers #1 and #18 carrying (RA3 coordinates 9837-10308 nt)
pJSB2.43 pUC18 with 61 bp oligonucleotides (primer #19 and #20), oligonucleotides are inserted in SmaI site in of pUC18
pJSB2.44 pJSB2.43 digested by NheI and EcoRI, blunt-ended with the use of Klenow fragment and self-ligated, to remove IRIV
pJSB2.55 pJSB2.44 oriT45ΔOM -lacI q -tacp-mobC-nic, BamHI-SalI fragment (lacI q -tacp-mobC-nic) from pMPB13.4
pJSB2.56 pJSB2.44 oriT45ΔOM -lacI q -tacp-nic, BamHI-SalI fragment (lacI q -tacp-nic) from pMPB13.3
pJSB2.57 pJSB2.55 oriT45ΔOM -lacI q -tacp-mobC-nic- TraRA3-korCp-korC, SmaI-SalI fragment (TraRA3-korCp-korC) from pJSB1.24
pJSB2.58 pJSB2.56 oriT45ΔOM -lacI q -tacp-nic- TraRA3-korCp-korC, SmaI-SalI fragment (TraRA3-korCp-korC) from pJSB1.24
pJSB4.1 pBBR1MCS-1 lacI q, tacp-mobC, BamHI-SalI from pJSB5.1
pJSB4.2 pBBR1MCS-1 lacI q, tacp-mobC1-129, BamHI-SalI from pJSB5.2
pJSB5.1 pGBT30 tacp-mobC, EcoRI-SalI fragment from pJSB6.1
pJSB5.2 pGBT30 tacp-mobC1-129, EcoRI-SalI fragment from pJSB6.2
pJSB6.1 pET28a T7p-mobC, EcoRI-SalI fragment from pJSB2.1
pJSB6.2 pET28a T7p-mobC1-129, EcoRI-SalI fragment from pJSB2.2,
pJSB6.30 pET28a T7p-mobC1-155, EcoRI-SalI fragment from pJSB2.30
pJSB7.9 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.9
pJSB7.10 pPT0I mobCp-xylE, 229 bp SphI-BamHI fragment PCR amplified with the use of primers #7 and #8 (RA3 coordinates 9623- 9852 nt)
pJSB7.11 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.11
pJSB7.12 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.12
pJSB7.13 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.13
pJSB7.14 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.14
pJSB7.15 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.15
pJSB7.16 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.16
pJSB7.17 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.17
pJSB7.18 pPT0I mobCp-xylE, SphI-BamHI fragment from pJSB2.18
pJSB8.1 pLKB4 cyaT18-mobC; EcoRI-HincII fragment from pJSB6.1 cloned between EcoRI-SmaI sites of pLKB4
pJSB8.30 pJSB8.1 digested by ClaI and self-ligated to remove 3′ end of mobC (mobC1-155)
pJSB9.1 pKGB4 mobC-cyaT18, EcoRI-SacI fragment amplified by PCR with primers #1 and #17
pJSB10.1 pLKB2 cyaT25-mobC; EcoRI-HincII fragment from pJSB5.1 cloned between EcoRI-SmaI sites of pLKB2
pJSB10.2 pLKB2 cyaT25-mobC1-129; EcoRI-HincII fragment from pJSB5.2 cloned between EcoRI-SmaI sites of pLKB2
pJSB11.1 pKGB5 mobC-cyaT25; EcoRI-SacI fragment from pJSB9.1.1
  1. 1BHR-broad-host-range.
  2. 2Institute of Biochemistry and Biophysics, Department of Microbial Biochemistry, Polish Academy of Sciences.
  3. 3Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, UK.