Measuring Staphylococcal Promoter Activities Using a Codon-Optimized β-Galactosidase Reporter.
Measuring Staphylococcal Promoter Activities Using a Codon-Optimized β-Galactosidase Reporter.
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DOI：
10.1007/978-1-0716-1550-8_6
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Bose JL
中科院分区：
文献类型：
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作者：
Krute CN;Seawell NA;Bose JL
lacZ基因和相应的β-半乳糖苷酶几十年来一直是细菌报告系统的支柱。我们已经使用这种多功能的报告分析表达谱从固体培养基上生长的菌株和肉汤培养。标准肉汤方案也可适用于96孔板，以允许在各种条件下高通量筛选启动子报告基因构建体。此外，对E. colilacZ基因能显著提高S.金黄色葡萄球菌，有利于提高灵敏度的筛选测定，检测低活性的启动子，并使用小的样品体积。在本章中，我们提供了标准和高通量定量分析的细节，我们经常使用的S。aureus转录谱分析。
The lacZ gene and corresponding β-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of β-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.