A Positive/Negative Selection Cassette for Red Recombination of BAC Clones

IF 1 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Applied Biochemistry and Microbiology Pub Date : 2024-06-10 DOI:10.1134/S0003683823602846
Y. Zhou, B. Xu, Z. Su, Z. Qin
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Abstract

Conventional gene cloning methods always require tedious processes confined by PCR conditions and restriction sites, which might result in a large number of redundant fragments in the constructs. These shortages can be overcome by using DNA recombination methods. Homologous recombination in Escherichia coli cells was used to develop positive/negative cassette for phage λ red operon recombination. In the bi-functional cassette, the Ala294Gly α-subunit mutant of phenylalanyl-tRNA synthetase (mPheS) was fused with the kanamycin-resistant (KanR) cassette under the control of the promoter of the E. coli gene NEOKAN as a single open reading frame (ORF) by PCR. KanR was utilized as a positive selection marker, while the mutant gene mPheS served as a negative selection marker. Two-step red-mediated recombination was used to replace the third exon of TMEM18 in the BAC clone RP23-25C13 with eGFP. Initially, the third TMEM18 exon in the BAC was replaced with the NEOKAN-mPheS-KanR cassette, and 40% of the clones on the positive selection plate (LB plate with kanamycin) were positive. The second step involved replacing the NEOKAN-mPheS-KanR cassette with eGFP in the modified BAC, and 60% of the clones on the negative plate (p-Cl-phe plate) were positive. Following the two steps, eGFP was used to replace the third exon of TMEM18 in the BAC. The NEOKAN-mPheS-KanR could be a promising choice as a positive/negative selection cassette for red recombination to establish constructs and make gene manipulation on BAC more applicable and effortless.

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用于 BAC 克隆红色重组的阳性/阴性选择盒
传统的基因克隆方法总是需要受 PCR 条件和限制位点限制的繁琐过程,这可能会导致构建体中出现大量冗余片段。利用 DNA 重组方法可以克服这些不足。我们利用大肠杆菌细胞中的同源重组技术开发了用于噬菌体λ红色操作子重组的正/负基因盒。在双功能盒中,苯丙氨酸-tRNA合成酶(mPheS)的Ala294Gly α-亚基突变体与卡那霉素抗性(KanR)盒在大肠杆菌基因NEOKAN启动子的控制下通过PCR融合成一个开放阅读框(ORF)。KanR 用作正选择标记,突变基因 mPheS 用作负选择标记。用两步红介导重组法将 BAC 克隆 RP23-25C13 中 TMEM18 的第三个外显子替换为 eGFP。首先,用 NEOKAN-mPheS-KanR 盒替换 BAC 中的第三个 TMEM18 外显子,阳性选择平板(含卡那霉素的 LB 平板)上 40% 的克隆呈阳性。第二步是将改良 BAC 中的 NEOKAN-mPheS-KanR 基因盒替换为 eGFP 基因盒,阴性选择板(p-Cl-phe 板)上 60% 的克隆呈阳性。在这两个步骤之后,用 eGFP 替代了 BAC 中 TMEM18 的第三个外显子。NEOKAN-mPheS-KanR可作为红色重组的正/负选择盒,用于建立构建体,使在BAC上的基因操作更适用、更简便。
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来源期刊
Applied Biochemistry and Microbiology
Applied Biochemistry and Microbiology 生物-生物工程与应用微生物
CiteScore
1.70
自引率
12.50%
发文量
75
审稿时长
6-12 weeks
期刊介绍: Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.
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