Table 5 Oligonucleotide primers used in this study Primer Sequence 5 ‘- 3′ F/cea7-BamHI GGATCCATGAGCGGTGGAGATGGACG R/cei7-PstI CTGCAGTCAGCCCTGTTTAAATCC
F/btuB-219-XbaI GGCTCTAGAAAACGGTGCCATCATACTTTG R/btuB+242-HindIII GGCAAGCTTATCATTGTAAAGCATCCACAATAG F/btuB-767 GTTCACCGTTGCTCGATACC R/btuB-1087 TCAGATAGATGCCGGTATTACG F/btuB-431-XbaI GCTCTAGAACGGGATTATTACGC F/btuB-671-XbaI GCTCTAGATCATCTCTTTCCC F/btuB-1043-XbaI GCTCTAGACCGCTGCGCGGA R/lacZ TTATTTTTGACACCAGACC F/gadA-176 GATCGCCCGAACAGCAA R/gadA+77 CGTGAATCGAGTAGTTC F/gadB-173 AATAACAGCATAAAACA R/gadB+77 CGTGAATCGAGTAGTTCC F/pal-XbaI TCTAGAGAGGCGTACAAGTTCTG R/pal-HindIII AAGCTTATCATTTCAATGATTCCTTTAC F/gadX-BamHI GGATCCATGCAACCACTACATGG MK-1775 molecular weight R/gadX-PstI CTGCAGCTATAATCTTATTCCTT F/gadX-393 TATACCGCTGCTTCTGAACG R/gadX-726 TCGCTCCTGATACTCTGTGG F/rrsA-483 CGTTACCCGCAGAAGAAGC R/rrsA-808 GTGGACTACCAGGGTATCTAATCC The underlined letters indicate QNZ mouse restriction sites. To assay btuB promoter activity, DNA fragments (461, 673, 913, and 1,285 bp) containing different portions selleck compound (Figure 3) of the btuB promoter was fused to lacZ. These fragments were generated by PCR using primers F/btuB-219-XbaI, F/btuB-431-XbaI,
F/btuB-671-XbaI, and F/btuB-1043-XbaI paired with the 3′ primer R/btuB +242-HindIII (Table 5). The resulting PCR products were digested with XbaI and HindIII and then inserted into corresponding sites on pKM005 that carries a promoterless lacZ gene , generating pKMbtuB461-lacZ, pKMbtuB673-lacZ, pKMbtuB913-lacZ, and pKMbtuB1285-lacZ. To mimic native expression of btuB, these btuB-lacZ fusions were transferred to the single copy plasmid vector pCC1 (Epicentre). The fragments containing btuB promoter and lacZ on pKM005 derivatives were amplified with primers F/btuB-219-XbaI, F/btuB-431-XbaI, F/btuB-671-XbaI, and F/btuB-1043-XbaI paired with the 3′ primer R/lacZ (Table 5), and the resulting 3.3, 3.5, 3.74, and 4.1-kb DNA fragments were separately PRKACG inserted into pGEM-TEasy (Promega) by TA cloning. The 3.3, 3.5, 3.74, and 4.1-kb fragments were then isolated from these pGEM-TEasy derivatives by NotI digestion and inserted into the NotI site of pCC1 vector, generating
pCB461lacZ, pCB673lacZ, pCB913lacZ, and pCB1285lacZ. The plasmid pC-lacZ that contains a promoterless lacZ gene inserted into pCC1 vector was used as a negative control. To produce GadX for DNA binding assay, pMalE-GadX that contains maltose-binding protein fused to GadX (MalE-GadX) was constructed. The 825-bp DNA fragment containing gadX was generated by PCR using pGadXY as the template and F/gadX-BamHI and R/gadX-PstI (Table 5) as primers and then ligated between the BamHI and PstI sites of pMAL-C2X (New England Biolab), resulting in pMalE-GadX. Production of ColE7 To produce the His6-tagged ColE7/ImE7 complex, E. coli strain XL1-Blue containing plasmid pQE30ColE7-Im7 was cultured in LB medium containing ampicillin (50 μg/ml) and tetracycline (20 μg/ml).