A vector for promoter trapping in Bacillus cereus
A vector for promoter trapping in Bacillus cereus
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DOI：
10.1016/s0378-1119(98)00544-7
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发表时间：
1999-01-21
期刊：
影响因子：
3.5
通讯作者：
Handelsman, J
中科院分区：
文献类型：
--
作者：
Dunn, AK;Handelsman, J
我们构建了一个启动子陷阱质粒，pAD 123，蜡样芽孢杆菌。该质粒含有无启动子的基因，其编码绿色荧光蛋白GFP mut 3a的突变形式，其被优化用于荧光激活的细胞分选[Cormack，B. P.，Valdivia，R.H.，Falkow，S.，1996.绿色荧光蛋白（GFP）的FAGS优化突变体。Gene 173，33-38.]。该质粒在大肠杆菌和B中复制并赋予耐药性。尾索我们在pAD 123中构建了一个包含29 000个含有尾丝虫B株UW 85染色体DNA的克隆的文库。在B中筛选一部分文库（988个克隆）的GFP表达。cereus UW 85的表达。通过用荧光成像仪目视检查来检测GFP表达。我们鉴定了21个在初始筛选中发荧光的克隆，并通过限制性酶切分析、测序和荧光强度的定量进一步表征了这些克隆。流式细胞术和细胞分选有效地分离了B。从10000倍过量的非表达细胞中获得表达GFP的蜡状细胞。选择的克隆提供了有用的标记来跟踪B。植物表面的蜡状菌种群。我们的研究结果表明，绿色荧光蛋白和pAD 123是有用的工具，用于确定在蜡状芽孢杆菌的调控序列，流式细胞术和细胞分选是一个有用的方法，用于筛选在这个载体中构建的大型库。(C)1999 Elsevier Science B. V.保留所有权利。
We constructed a promoter-trap plasmid, pAD123, for Bacillus cereus. This plasmid contains a promoterless gene that encodes a mutant version of the green fluorescent protein, GFPmut3a, that is optimized for fluorescence-activated cell sorting [Cormack, B.P., Valdivia, R.H., Falkow, S., 1996. FAGS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-38.]. The plasmid replicates and confers drug resistance in both Escherichia coli and B. cercus. We constructed a library in pAD123, which consists of 29 000 clones containing chromosomal DNA from B, cercus strain UW85. A portion of the library (988 clones) was screened for GFP expression in B. cereus UW85 using a 96-well microtiter dish assay. GFP expression was detected by visual inspection with a fluorimager. We identified 21 clones as fluorescing in the initial screen, and further characterized these clones by restriction analysis, sequencing, and quantification of fluorescence intensity. Flow cytometry and cell sorting efficiently separated B. cereus cells expressing GFP from a 10 000-fold excess of non-expressing cells. Selected clones provided useful markers to follow B. cereus populations on plant surfaces. Our results indicate that GFP and pAD123 are useful tools for identifying regulatory sequences in Bacillus cereus, and that flow cytometry and cell sorting is a useful method for screening large libraries constructed in this vector. (C) 1999 Elsevier Science B.V. All rights reserved.