A vector for promoter trapping in Bacillus cereus |
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| Institution: | 1. University of Wisconsin-Madison, Department of Plant Pathology, 1630 Linden Dr., Madison, WI 53706, USA;2. University of Wisconsin-Madison, Department of Bacteriology, Madison, WI 53706, USA;1. Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Jinnan District, Tianjin, 300350, PR China;2. Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Jinnan District, Tianjin, 300350, PR China;3. Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Science, Tianjin Normal University, Binshuixi Road 393, Xiqing District, Tianjin 300387, PR China;1. Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, School of Life Science, Huaiyin Normal University, No. 111, West Changjiang Road, Huai’an, China;2. Agro-Tech Extension and Service Center, No. 55, West Jiankang Road, Huai’an, China;3. Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huai’an, China;1. College of Life Sciences, Northeast Forestry University, Harbin, China;2. Key Laboratory for Enzyme and Enzyme-like Material Engineering of Heilongjiang, Harbin, China;1. Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, 210095, PR China;2. Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China |
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| Abstract: | 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. FACS-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. cereus. We constructed a library in pAD123, which consists of 29 000 clones containing chromosomal DNA from B. cereus 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. |
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