Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland.
York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK.
Microbiology (Reading). 2010 Jun;156(Pt 6):1600-1608. doi: 10.1099/mic.0.035758-0. Epub 2010 Feb 11.
Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis.
质粒 pBaSysBioII 被构建用于高通量分析枯草芽孢杆菌中的基因表达。它是一种整合型质粒，带有连接酶独立克隆（LIC）位点，允许生成带有所需启动子的转录 gfpmut3 融合物。整合是通过 Campbell 型事件发生的，是非诱变的，将融合物定位在同源染色体位置上。使用对磷酸盐可用性、生长速率和碳源有响应的 phoA、murAA、gapB、ptsG 和 cggR 启动子，我们表明可以建立启动子活性的详细图谱，并且可以在刺激后 1 分钟内测量到对变化条件的响应。这使得 pBaSysBioII 成为一种非常通用的工具，可用于实时分析枯草芽孢杆菌生长细胞中的基因表达。
