We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

中文翻译：

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I-Block：一种基于大肠杆菌的简单测定法，用于研究蛋白质的序列特异性DNA结合。

我们已经开发出一种简单的方法，称为I-Block分析，该方法可以检测大肠杆菌中蛋白质与DNA的序列特异性结合。该方法通过检测目标蛋白质和RNA聚合酶之间竞争与质粒载lacI启动子变体中重叠目标位点的结合而起作用。该测定法利用两个质粒和一个大肠杆菌宿主菌株，其中的Lac阻遏物（lacI）基因已被删除。质粒之一携带具有在lacI启动子中产生的独特的NheI限制性位点的lacI基因。被测蛋白质的潜在识别序列被插入NheI位点。将质粒引入大肠杆菌ΔlacI宿主可抑制宿主细菌的组成型β-半乳糖苷酶合成。如果从兼容质粒表达的研究蛋白与lacI启动子中的靶位点结合，它将干扰lacI转录并导致β-半乳糖苷酶活性增加。用两种锌指蛋白，λ噬菌体cI857阻遏物和靶向lacI启动子的CRISPR-dCas9测试了该方法。已显示出I-Block分析可与标准液体培养物一起使用，培养物在微孔板中生长，并且菌落在X-gal指示板上。