Multiple genes deletion based on Cre-loxP marker-less gene deletion system for the strains from the genus of Pectobacterium.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2024-12-01 Epub Date: 2024-07-31 DOI:10.1007/s10529-024-03518-8

Shu Che, Yuanxu Zhuo, Liping Yang, Huan Wang, Zhongli Cui, Jiaqin Fan

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引用次数: 0

Abstract

Objective: To introduce the Cre-loxP system for constructing marker-less multiple-gene deletion mutants in Pectobacterium, overcoming limitations of antibiotic markers and enhancing the understanding of pathogenic mechanisms.

Results: Firstly, a plasmid named pEX18-Cre, containing a sacB sucrose suicide gene, was constructed to express Cre recombinase in Pectobacterium. Secondly, a mutant in which the loxP-Km fragment replaced the target gene was obtained through homologous recombination double-crossover with the chromosome. Finally, pEX18-Cre was introduced into the mutant to excise the DNA between the loxP sites, thereby removing the markers and achieving multiple gene deletions. By utilizing the Cre-loxP system, we successfully constructed multiple marker-less gene deletion mutants in Pectobacterium strains.

Conclusions: The Cre-loxP system efficiently creates marker-less multiple-gene deletion mutants, enhancing the study of Pectobacterium pathogenic mechanisms by overcoming antibiotic marker limitations.

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基于 Cre-loxP 无标记基因缺失系统的果胶杆菌属菌株的多基因缺失。

目的：引入 Cre-loxP 系统，构建无标记的果胶杆菌多基因缺失突变体：引入 Cre-loxP 系统构建无标记的果胶杆菌多基因缺失突变体，克服抗生素标记的局限性，提高对致病机制的认识：结果：首先，构建了含有 sacB sucrose 自杀基因的质粒 pEX18-Cre，用于在果胶杆菌中表达 Cre 重组酶。其次，通过与染色体的同源重组双交叉，获得了loxP-Km片段取代目的基因的突变体。最后，将 pEX18-Cre 导入突变体，切除 loxP 位点之间的 DNA，从而去除标记，实现多基因缺失。通过利用 Cre-loxP 系统，我们成功地在果胶杆菌菌株中构建了多个无标记基因缺失突变体：结论：Cre-loxP 系统能有效地创建无标记的多基因缺失突变体，克服了抗生素标记的限制，从而加强了对果胶杆菌致病机制的研究。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.