原创力文档a simple method for construction of pir+ enterobacterial hosts for maintenance of r6k replicon plasmids一个简单的方法建设pir + enterobacterial主机的维护r6k复制子质粒.pdfVIP
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asimplemethodforconstructionofpirenterobacterialhostsformaintenanceofr6krepliconplasmids一个简单的方法建设pirenterobacterial主机的维护r6k复制子质粒
Kvitko et al. BMC Research Notes 2012, 5:157
/1756-0500/5/157
TECHNICAL NOTE Open Access
A simple method for construction of pir+
Enterobacterial hosts for maintenance of R6K
replicon plasmids
1† 1† 1 1 1 1
Brian H Kvitko , Steven Bruckbauer , John Prucha , Ian McMillan , Erin J Breland , Stephanie Lehman ,
1 2 1 1*
Katie Mladinich , Kyoung-Hee Choi , RoxAnn Karkhoff-Schweizer and Herbert P Schweizer
Abstract
Background: The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K
plasmid and derivatives harboring its g origin of replication (oriR6Kg) is dependent on the pir gene-encoded π
protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support
replication of plasmids harboring oriR6Kg. In Escherichia coli this is commonly achieved by chromosomal integration
of pir either via lysogenization with a lpir phage or homologous recombination at a pre-determined locus.
Findings: Current methods for construction of host strains for oriR6Kg-containing plasmids involve procedures that
do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps.
In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using
a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and
mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition,
we isolated pir+ and pir-116 expr
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