کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2090037 | 1081467 | 2013 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis](/preview/png/2090037.png)
• We use mini Zeocin resistance gene as positive-selectable marker.
• Strictly controlled xyl promoter from the Bacillus subtilis replaced non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium.
• The frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%.
• The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3 days.
There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3 days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.
Journal: Journal of Microbiological Methods - Volume 95, Issue 2, November 2013, Pages 207–214