Conditional-replication, integration, excision, and retrieval plasmid-hostsystems for gene structure-function studies of bacteria

We have developed a series of powerful and versatile conditional-replicatio n, integration, and modular (CRIM). plasmids. CRIM plasmids can be replicat ed at medium or high copy numbers in different hosts for making gene (or mu tant), libraries. They can be integrated in single copies into the chromoso mes of Escherichia coli and related bacteria to study gene function under n ormal physiological conditions. They can be excised from the chromosome, e. g.,, to, verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombin ation at one of five different phage attachment sites. Integrants are selec ted as, antibiotic-resistant transformations. Since CRIM plasmids encode di fferent forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from dive rse sources.. Following integration, integrants are stably maintained in th e absence of antibiotic selection., Each, CRIM plasmid has, a polylinker or one of several promoters for ectopic expression of the inserted DNA.. Thei r modular design allows, easy construction of new variants with different c ombinations of features. We also. report a series of easily. curable, low-c opy-number helper plasmids encoding all the requisite Int proteins, alone o r with the respective Xis protein. These helper plasmids facilitate integra tion, excision ("curing"), or retrieval of the CRM plasmids.