Construction of Versatile Cloning Vectors

In all vectors, the PstI site in the bla gene was removed by site-directed mutagenesis using the oligonucleotides TM2206 and TM2207. The resulting vector is marked with the auxiliary “blamut”. Primer design and mutagenesis were performed according to the manufacturer’s instructions for the QuikChange® II Site-Directed Mutagenesis Kit (Agilent Technologies). All plasmids were test-digested to confirm removal of the restriction site.
To create the empty vector pBS1C, pDG1662blamut was cut with PstI to remove one XbaI site and the spc^r outside of the integrative part. The 6 kb fragment was religated. The remaining PstI site was mutated via site-directed mutagenesis with the primers TM2845 and TM2846. To insert the MCS, the vector was cut with EcoRI. The MCS was amplified by PCR from pSB1C3 with the Primers TM2843 and TM2844, cut with EcoRI and BsaI (EcoRI-compatible overhang) and ligated into the vector. The remaining NgoMIV sites were removed by subsequent site-directed mutagenesis with TM3005 + TM3006, TM3011 + TM3012 and TM3013 + TM3014, respectively, resulting in pBS1C.
To create the empty vector pBS2E, pAX01 was cut with SacI. The 6.3 kb fragment was religated to remove the xylR-P_xylA-fragment. The PstI site in bla was removed. The vector was cut with XbaI and the 6 kb fragment religated to reduce the amount of forbidden restriction sites. The lost terminator of erm was replaced by the PCR-amplified terminator with BsaI-overhangs (primers: TM2975 and TM2976). The vector was opened with XbaI and ligated with the PCR product cut with BsaI (XbaI-compatible overhang). The correct direction was checked by sequencing and removal of the XbaI-site was confirmed. Finally, the MCS was amplified from pSB1C3 (TM2608 and TM2609) with PstI and NsiI overhangs, cut with PstI and NsiI and ligated into the PstI-cut vector. Correct orientation of the insert was confirmed by restriction digest and sequencing. The remaining NgoMIV sites were removed by subsequent site-directed mutagenesis with TM3011 + TM3012 and TM3028 + TM3029, respectively, resulting in pBS2E.
To create the empty vector pBS4S, the erm-resistance outside of the integrative part of pDG1731 was removed via cut-ligation with MluI and BssHI, resulting in a 4.7 kb vector. The PstI site in bla was removed as described above. The PstI site in thrB was removed performing site-directed mutagenesis with the primers TM2835 and TM2836. The spc-promoter with upstream PstI-overhang was amplified with the primers TM2837 and TM2838 and cut with PstI and PciI. The MCS was cut from pSB1C3 with EcoRI and PstI. The vector was cut with EcoRI and PciI and the 4.3 kb-fragment ligated with both cut DNA-fragments. The remaining NgoMIV sites were removed by subsequent site-directed mutagenesis with TM3005 + TM3006 and TM3011 + TM3012, respectively, resulting in pBS4S.
To create the reporter vector pBS1ClacZ, pAC6blamut was cut with EcoRI and PstI. The primers TM2301 and TM2302 (1 pM) were mixed, heated (95°C, 10 min), re-annealed (50°C, 10 min) to become double stranded with 4 bp overhangs and ligated into the vector. The vector was cut with EcoRI and PstI and ligated with the MCS from pSB1C3 cut with EcoRI and PstI. A PstI site in the E. coli ori was removed by cutting with BglII and religation of the 11 kb fragment.
To create the reporter vector pBS3Clux, the PstI site in the sacA locus of pAH328blamut was removed by site-directed mutagenesis with the primers TM2885 and TM2886. Then, the XbaI site of luxD was removed similarly with the Primers TM2887 and TM2888. The MCS was amplified by PCR from pSB1C3 with the primers TM2843 and TM2884, cut with EcoRI and BsaI (SpeI-overhang) and ligated into the EcoRI/SpeI-cut vector.