کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2091729 1545951 2007 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Targeting the Bacillus subtilis genome: An efficient and clean method for gene disruption
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Targeting the Bacillus subtilis genome: An efficient and clean method for gene disruption
چکیده انگلیسی

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented β cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne β site-specific recombinase is transferred into the background. Protein β catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 70, Issue 3, September 2007, Pages 389–394
نویسندگان
, , ,