{"title":"Optimizing CaCl<sub>2</sub>-mediated transformation of Pseudomonas aeruginosa SDK-6 with pJN105 using OFAT: A novel and efficient cloning approach.","authors":"Damanjeet Kaur, Vijay Singh, Saurabh Gupta","doi":"10.1007/s00294-024-01295-5","DOIUrl":null,"url":null,"abstract":"<p><p>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<sup>5</sup> 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.</p>","PeriodicalId":10918,"journal":{"name":"Current Genetics","volume":null,"pages":null},"PeriodicalIF":1.8000,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Genetics","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s00294-024-01295-5","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
引用次数: 0
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 × 105 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.
期刊介绍：
Current Genetics publishes genetic, genomic, molecular and systems-level analysis of eukaryotic and prokaryotic microorganisms and cell organelles. All articles are peer-reviewed. The journal welcomes submissions employing any type of research approach, be it analytical (aiming at a better understanding), applied (aiming at practical applications), synthetic or theoretical.
Current Genetics no longer accepts manuscripts describing the genome sequence of mitochondria/chloroplast of a small number of species. Manuscripts covering sequence comparisons and analyses that include a large number of species will still be considered.
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