TABLE 2

Key plasmids and relevant derivatives of these plasmids used for the construction of A. vinelandii manipulated strains

PlasmidaRelevant gene(s) cloned or plasmid manipulationbParent vectorSource or reference
pEB001Plasmid containing Mariner transposon and transposase24
pBB052pUC19 with Kanr from pUC4K in place of AmprpUC1950
pBB053Removed NdeI site from pUC19 by silent mutationpUC1925
pBB073Moved spectinomycin/streptomycin resistance cassette from pHP45Ω into EcoRI site of pBB052pBB05251
pBBTET3pUC19 with Tetr in place of AmprpUC1925
pPCRKAN4Cloned Kan cassette from pBBR1MCS-2 into pBBTET3pBBTET325
pLACZF12Cloned lacZ gene from E. coli into pBBTET3 and removed various restriction sites by silent mutation with site-specific mutagenesispBBTET3This study
pPCRSCRK2Cloned scrX gene and flanking regions from A. vinelandii into pBB053pBB053This study
pPCRSCRK5Removed restriction sites from pPCRSCRK2 by blunt fill-in; performed PCR to remove scrX gene from plasmidpBB053This study
pPCRSCRK7Performed additional blunt fill-in and site-specific mutagenesis to remove additional restriction sitespBB053This study
pPCRSCRK28Moved Kan cassette from pPCRKAN4 into pPCRSCRK7, then removed restriction sites by site-specific mutagenesis with silent mutationspBB053This study
pPCRSCRK31*Moved lacZ gene from pLACZF12 into pPCRSCRK28pBB053This study
pPCRNH3-10Cloned nifA gene from A. vinelandii into pBB114pBB114This study
pPCRNH3-11Cloned nifLA genes and flanking regions from A. vinelandii into pBB053pBB053This study
pPCRNH3-12Removed restriction site from pPCRNH3-11 by blunt fill-inpBB053This study
pPCRNH3-13Performed PCR to remove nifLA genes from pPCRNH3-12 and add XbaI and BamHI sitespBB053This study
pPCRNH3-14*Moved nifA gene from pPCRNH3-10 into pPCRNH3-13pBB053This study
pPCRNH3-15*Moved Str cassette from pBB073 into pPCRNH3-13pBB053This study
pPCRNH3-21*Moved Tet cassette pBBTET3 into pPCRNH3-13pBB053This study
pPCRNH3-42*Removed segment of nifL gene from pPCRNH3-11 and inserted Kan cassette from pPCRKAN4 similar to approach taken to construct pBB369pBB0538; this study
pPCRNH3-43*Removed larger segment of nifL gene from pPCRNH3-11 than was done in pPCRNH3-42 and inserted Kan cassette from pPCRKAN4 slightly further upstream of nifA as shown in Fig. 7pBB053This study
pPCRURE1Cloned ureABC genes and flanking regions from A. vinelandii into pUC19pUC19This study
pPCRURE2Performed PCR to remove ureABC genes from pPCRURE1pUC19This study
pPCRURE3*Moved Str cassette pBB073 into pPCRURE2pUC19This study
pPCRAMTBK1Cloned gene amtB and flanking regions from A. vinelandii into pBB053pBB053This study
pPCRAMTBK2Performed PCR to remove gene amtB from pPCRAMTBK1pBB053This study
pPCRAMTBK3*Moved Kan cassette from pPCRKAN4 into pPCRAMTBK2pBB053This study
  • a The sequences of all plasmids in this study are available upon request. Plasmids indicated by an asterisk (*) are completed vectors that were used to transform A. vinelandii.

  • b Tetr, tetracycline resistance; Ampr, ampicillin resistance; Strr, streptomycin resistance; Kanr, kanamycin resistance.