用 OFAT 优化 Pseudomonas aeruginosa SDK-6 用 pJN105 的 CaCl2 介导转化：一种新颖有效的克隆方法。

Optimizing CaCl-mediated transformation of Pseudomonas aeruginosa SDK-6 with pJN105 using OFAT: A novel and efficient cloning approach.

机构信息

Department of Microbiology, Mata Gujri College, Fatehgarh Sahib, Punjab, 140406, India.

Department of Biotechnology and Food Technology, Punjabi University, Patiala, Punjab, 147002, India.

出版信息

Curr Genet. 2024 Jul 31;70(1):11. doi: 10.1007/s00294-024-01295-5.

DOI:10.1007/s00294-024-01295-5

PMID:39083080

Abstract

Cloning and expression of a gene in the desired host is required for optimum production in recombinant strains. The present research is the first attempt to optimize the physiological conditions for the transformation of Pseudomonas aeruginosa SDK-6 with pJN105. Different factors, such as inoculum size, incubation period, heat shock temperature, and heat shock time were optimized using one factor at a time (OFAT) followed by the selection of transformants using gentamicin resistance marker. The maximum number of transformants (2.002 ± 0.077 × 10 cfu/ µg of plasmid DNA) were reported with 0.5% (v/v) inoculum, an incubation period of 3 h, and heat shock treatment at 50 °C for 1 min. An overall 12-fold increase in transformation efficiency was observed. The presence of a 6055 bp band on agarose gel confirmed the transformation of Pseudomonas aeruginosa with the vector pJN105.

摘要

为了在重组菌株中实现最佳生产，需要在所期望的宿主中克隆和表达目的基因。本研究首次尝试优化 Pseudomonas aeruginosa SDK-6 用 pJN105 进行转化的生理条件。使用逐个因素优化（OFAT）优化了不同因素，如接种量、孵育时间、热激温度和热激时间，然后使用庆大霉素抗性标记物选择转化体。报道的最大转化体数量（2.002 ± 0.077 × 10 cfu/µg 质粒 DNA）是在 0.5%（v/v）接种量、孵育时间 3 小时和 50°C 热激处理 1 分钟的条件下获得的。转化效率总体提高了 12 倍。琼脂糖凝胶上存在 6055 bp 条带证实了 Pseudomonas aeruginosa 与载体 pJN105 的转化。