One-step cloning and targeted duplication of Pantoea ananatis chromosomal fragments

IF 1.7 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Journal of microbiological methods Pub Date : 2024-07-19 DOI:10.1016/j.mimet.2024.106999

O. Igonina, V. Samsonov, N. Stoynova

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Abstract

In this study, we describe a novel method for one-step cloning and targeted duplication of P. ananatis chromosomal fragments. According to this method, the chromosomal region of interest is subcloned in vivo via λ Red recombination into the short synthetic non-replicable DNA fragment containing the excisable antibiotic-resistance marker gene and φ80 att-P site. The resulting circular non-replicating DNA molecule was immediately inserted into an alternative chromosomal locus due to φ80-integrase activity. To this end, the specially designed helper plasmid pONI, which can provide both the λ Red recombineering and φ80-integrase-mediated insertion, was constructed. In the described method, PCR amplification of the cloning fragment is unnecessary, making it convenient for manipulation of long-length DNA. Additionally, the possibility of spontaneous mutations occurring is completely precluded. This method was effectively used for the targeted chromosomal integration of additional copies of individual genes and operons up to 16 kb in size.

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盘尾丝菌染色体片段的一步克隆和定向复制。

在本研究中，我们描述了一种一步克隆和定向复制 P. ananatis 染色体片段的新方法。根据该方法，感兴趣的染色体区域在体内通过 λ Red 重组亚克隆到含有可切除抗生素标记基因和 φ80 att-P 位点的短合成不可复制 DNA 片段中。由于φ80整合酶的活性，由此产生的环状不可复制DNA分子立即被插入到另一个染色体位点上。为此，我们构建了专门设计的辅助质粒 pONI，它既能提供 λ Red 重组，也能提供φ80-整合酶介导的插入。在所述方法中，无需对克隆片段进行 PCR 扩增，因此便于操作长 DNA。此外，还完全排除了发生自发突变的可能性。这种方法可有效地用于对单个基因和操作子的额外拷贝进行有针对性的染色体整合，拷贝大小可达 16 kb。

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来源期刊

Journal of microbiological methods 生物-生化研究方法

CiteScore

4.30

自引率

4.50%

发文量

151

审稿时长

29 days

期刊介绍： The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach. All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.