کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6128468 | 1592822 | 2016 | 36 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species
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موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
میکروب شناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 Ã 10â3 for B. fragilis and 1.7 Ã 10â3 for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Anaerobe - Volume 42, December 2016, Pages 81-88
Journal: Anaerobe - Volume 42, December 2016, Pages 81-88
نویسندگان
Yasuhiro Kino, Haruyuki Nakayama-Imaohji, Masashi Fujita, Ayano Tada, Saori Yoneda, Kazuya Murakami, Masahito Hashimoto, Tetsuya Hayashi, Katsuichiro Okazaki, Tomomi Kuwahara,