| DOI | Resolve DOI: https://doi.org/10.1016/S0378-1097(00)00463-8 |
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| Author | Search for: Figueira, Marianne M.1; Search for: Laramée, Louise1; Search for: Murrell, J. Colin; Search for: Groleau, Denis1; Search for: Miguez, Carlos B.1 |
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| Affiliation | - National Research Council of Canada. NRC Biotechnology Research Institute
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| Format | Text, Article |
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| Subject | biotechnology; biosynthesis; Copper; Culture Media; Electroporation; Gene Expression Regulation,Bacterial; Genetic Vectors; genetics; Green fluorescent protein expression; growth & development; Lac Operon; Luminescent Proteins; metabolism; methods; Methylobacterium; Methylobacterium extorquens; Methylotroph; Oxygenases; Plasmids; Promoter Regions (Genetics); Proteins; Recombinant Proteins; Support,Non-U.S.Gov't; Transformation,Bacterial |
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| Abstract | The production of green fluorescent protein (GFP) in Methylobacterium extorquens was studied by creating four different constructs using pJB3KmD, pRK310 and pVK101 vectors, as well as pLac and soluble methane monooxygenase (sMMO) promoters. Plasmids were introduced into the cells by electroporation. Expression of GFP by selected clones was evaluated by growing cells in complex or defined media. The use of pRK310 as an expression vector containing the lacZ promoter resulted in a 100-fold increase of GFP production when compared to cells containing the pLac-GFP-pJB3KmD construct. Higher production of GFP was observed also in cells containing pLac- GFP-pRK310 and pmmoX-GFP-pVK101 constructs. While the transcriptional regulation of the smmo gene in Methylosinus trichosporium OB3b is known to be copper-dependent, expression of GFP by M. extorquens clones harboring pmmoX-promoters was not strongly controlled by the presence of copper in the medium. The production of GFP was generally constant throughout the growth of M. extorquens carrying the pLac-GFP-pRK310 construct. GFP yields varied between 850 and 1000 Wg of GFP g biomass⁻¹. However, the yield of GFP in cells carrying pmmoX-GFP-pVK101 was somewhat reduced after the mid-exponential phase of growth. |
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| Publication date | 2000 |
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| In | |
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| Note | UI - 20564155LA - engRN - 0 (Culture Media)RN - 0 (Genetic Vectors)RN - 0 (Luminescent Proteins)RN - 0 (Plasmids)RN - 0 (Recombinant Proteins)RN - 147336-22-9 (green fluorescent protein)RN - 7440-50-8 (Copper)RN - EC 1.13. (Oxygenases)RN - EC 1.14.13.25 (methane monooxygenase)PT - Journal ArticleDA - 20010104IS - 0378-1097SB - IMCY - NETHERLANDSJC - FML |
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| Language | English |
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| NRC number | 37693 |
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| NPARC number | 3539810 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 958f3a7b-e3d8-4f34-ac89-d9bb0ac6dbfb |
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| Record created | 2009-03-01 |
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| Record modified | 2020-03-26 |
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