Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria - PubMed (original) (raw)

FIG. 1

Structures of CRIM plasmid series. Gene designations include aadA (aminoglycoside adenyltransferase for spectinomycin and streptomycin resistance), bla (β-lactamase for ampicillin resistance), cat (chloramphenicol acetyltransferase), dhfr (dihydrofolate reductase for trimethoprim resistance), gen (gentamicin-3-acetyltransferase for gentamicin resistance), kan (aminoglycoside 3′-phosphotransferase for kanamycin resistance), _pstS_∗ (a mutant pstS, Pi-specific binding protein), tetA (tetracycline resistance), and uidAf (the uidA2 fusion in pSK49Δ::uidA2 [16]). The multiple cloning site (MCS) is from pUC18 (44). Unique sites within the MCS of pAH68 include, from left to right, _Sph_I, _Pst_I, _Sal_I, _Xba_I, _Bam_HI, _Sma_I, _Kpn_I, _Sac_I, and _Eco_RI. All sites are illustrated for the enzymes shown. Sites destroyed during construction are marked with an asterisk. Modules are flanked by _Sph_I, _Eco_RI (_Bam_HI or _Nde_I), _Nhe_I, _Nco_I, _Not_I, and _Cla_I (and/or _Bsa_I) sites. Plasmids with aadA, bla, or gen facilitate certain constructions as they have a unique _Nco_I site. Due to the manner in which these plasmids were constructed, the P araB region of pAH150, but not of pLA2, encodes an N-terminal portion of AraC as a fusion protein. As a consequence, P araB is expressed at a normal level in pLA2 but at a much reduced level in pAH150 (see the text). All attP sites were designed taking into account information on important DNA binding sites and structure (23, 35, 43). Accordingly, the _att_P21 sequence encodes the C terminus of icd and the _att_P22 sequence includes sequences for the thrW tRNA gene (6). Unexpectedly, we found that CRIM plasmids carrying _att_P22s or _att_φ80s (Table 3) failed to integrate or gave very few integrants, respectively, suggesting that additional att sequences are required (data not shown). Plasmids carrying a longer _att_φ80 site (such as pAH153 and pAH162) integrated efficiently, while those carrying the longer _att_P22 site (such as pAH154) integrated less frequently than others (see the text). Primers routinely used to verify cloned inserts by PCR or DNA sequencing include rgnB-f (TTGTCGGTGAACGCTCTCCT), ParaB-f (CACATTGATTATTTGCACGG), PrhaB-f (CGTTCATCTTTCCCTGGT), and tL3-r (AGGATGCGTCATCGCCATTA). Priming sites rgnB-f and tL3 are common to all CRIM plasmids and are useful for sequencing inserts in all CRIM plasmids, except those containing _lacI_q, in which only tL3-r remains useful.