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Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath
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Ali, Hanif and Murrell, J. C. (J. Colin). (2009) Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath. Microbiology, Vol.155 (No.3). pp. 761-771. ISSN 1350-0872
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Official URL: http://dx.doi.org/10.1099/mic.0.021816-0
Abstract
A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XyIE) or beta-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) sigma(54) promoter or particulate methane monooxygenase (pMMO) sigma(70) promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When beta-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QR Microbiology | ||||
| Divisions: | Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010) | ||||
| Journal or Publication Title: | Microbiology | ||||
| Publisher: | Society for General Microbiology | ||||
| ISSN: | 1350-0872 | ||||
| Official Date: | March 2009 | ||||
| Dates: |
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| Volume: | Vol.155 | ||||
| Number: | No.3 | ||||
| Number of Pages: | 11 | ||||
| Page Range: | pp. 761-771 | ||||
| Identification Number: | 10.1099/mic.0.021816-0 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Restricted or Subscription Access | ||||
| Funder: | Natural Environment Research Council | ||||
| Grant number: | NER/A/S/2002/00876 | ||||
| URI: | http://wrap.warwick.ac.uk/id/eprint/28244 |
Data sourced from Thomson Reuters' Web of Knowledge
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