Wong Julia L, Vogt Stefanie L, Raivio Tracy L
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada.
Methods Mol Biol. 2013;966:337-57. doi: 10.1007/978-1-62703-245-2_21.
We describe methods for screening the E. coliASKA overexpression library for clones that lead to altered expression of reporter genes. First, a promoter of interest is cloned upstream of either the lacZor luxCDABEgenes to yield reporter genes in which transcription is proportional to the levels of β-galactosidase or luminescence produced by strains carrying the reporter. The ASKA library is then condensed into two 96-well plates resulting in mixed preparations of 12 plasmids in each well. The plasmids in each well are transformed into the reporter strain and transformants are screened for either altered β-galactosidase or light production. The genes contained in ASKA clones that result in altered reporter gene expression are amplified and sequenced and the ASKA clone for the gene identified is retransformed into the parent reporter strain to confirm the effect. We have used screens like this one to look for new E. coligenes that, when over-expressed, result in the altered expression of promoters that are regulated by the envelope stress response. The identity of the clones can yield information about the nature of inducing cues and/or additional regulatory molecules. The techniques are broadly applicable to any microbial function of interest.
我们描述了从大肠杆菌ASKA过表达文库中筛选可导致报告基因表达改变的克隆的方法。首先，将感兴趣的启动子克隆到lacZ或luxCDABE基因的上游，以产生报告基因，其中转录与携带该报告基因的菌株产生的β-半乳糖苷酶水平或发光水平成正比。然后将ASKA文库浓缩到两个96孔板中，每个孔中得到12种质粒的混合制剂。将每个孔中的质粒转化到报告菌株中，并筛选转化体中β-半乳糖苷酶或发光的改变情况。对导致报告基因表达改变的ASKA克隆中所含的基因进行扩增和测序，并将鉴定出的该基因的ASKA克隆重新转化到亲本报告菌株中以确认其作用。我们已经使用了这样的筛选方法来寻找新的大肠杆菌基因，这些基因在过表达时会导致受包膜应激反应调控的启动子表达改变。克隆的身份可以提供有关诱导信号和/或其他调节分子性质的信息。这些技术广泛适用于任何感兴趣的微生物功能。
