a single copy integration vector that integrates at an engineered site on the staphylococcus aureus chromosome集成单拷贝向量集成在一个设计网站的金黄色葡萄球菌染色体.pdfVIP

Lei et al. BMC Research Notes 2012, 5:5 /1756-0500/5/5 SHORT REPORT Open Access A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome * Mei G Lei, David Cue, Jimena Alba, Jennifer Junecko, Justin W Graham and Chia Y Lee Abstract Background: Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. Findings: We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We