The Experts below are selected from a list of 678 Experts worldwide ranked by ideXlab platform
Hermann Bujard - One of the best experts on this subject based on the ideXlab platform.
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Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR/O, the TetR/O and AraC/I1-I2 Regulatory Elements
Nucleic Acids Research, 1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.
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independent and tight regulation of transcriptional units in escherichia coli via the lacr o the tetr o and arac i1 i2 regulatory elements
Nucleic Acids Research, 1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.
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Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements
1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of P lac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd g..
S A Dassie - One of the best experts on this subject based on the ideXlab platform.
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binding of the highly toxic tetracycline derivative Anhydrotetracycline to bovine serum albumin
Biological & Pharmaceutical Bulletin, 2011Co-Authors: Martha Ines Burgos, Ricardo Fernandez, Maria Soledad Celej, Laura I Rossi, Gerardo D Fidelio, S A DassieAbstract:Tetracycline (TC) derivatives are extensively used as antibiotics in human and animal medicine and, very recently, they have been screened as anti-amyloidogenic drugs. Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug. We evaluated the interaction of AHTC with bovine serum albumin (BSA), one of the main carriers of amphiphilic molecules in blood, using three complementary analytical methods: fluorescence spectroscopy, isothermal titration calorimetry and differential scanning calorimetry. AHTC bound to BSA with an association constant in the order of 10(5) M(-1). Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.
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electroanalytical procedure to resolve a sample solution containing tetracycline and its toxic degraded product Anhydrotetracycline
Journal of Electroanalytical Chemistry, 2008Co-Authors: Ricardo Fernandez, S A DassieAbstract:Abstract The difference in the hydrophobicity of tetracycline and Anhydrotetracycline was used to quantify each compound in a mixture. Cyclic voltammetry was employed to characterize the charge transfer processes for tetracycline and Anhydrotetracycline across the H2O|1,2-dicloroethane interface. In this paper, we present a comprehensive and detailed study that includes the transfer potential dependence of pH value, the elucidation of the mechanism of transfer using mechanical control of convective flux (N. Wilke, R.A. Iglesias, S.G. Chesniuk, S.A. Dassie, A.M. Baruzzi, Bull. Chem. Soc. Jpn. 75 (2002) 235), and the determination of partition coefficient values employing the change of voltammetric current with external parameters (J.I. Garcia, R.A. Fernandez, A.J. Ruggeri, S.A. Dassie, J. Electroanal. Chem. 594 (2006) 80). These studies allow us to introduce an electroanalytical procedure to resolve different mixtures containing tetracycline and Anhydrotetracycline in acid degraded tetracycline samples.
Sabine Ehrt - One of the best experts on this subject based on the ideXlab platform.
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inactivation of fructose 1 6 bisphosphate aldolase prevents optimal co catabolism of glycolytic and gluconeogenic carbon substrates in mycobacterium tuberculosis
PLOS Pathogens, 2014Co-Authors: Susan Puckett, Dirk Schnappinger, Carolina Trujillo, Hyungjin Eoh, Joeli Marrero, John S Spencer, Mary Jackson, Kyu Y Rhee, Sabine EhrtAbstract:Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by Anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.
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doi:10.1093/nar/gkp015 Improved tetracycline repressors for gene silencing in mycobacteria
2008Co-Authors: Marcus Klotzsche, Sabine EhrtAbstract:Tetracycline repressor (TetR)-controlled expres-sion systems have recently been developed for mycobacteria and proven useful for the construc-tion of conditional knockdown mutants and their analysis in vitro and during infections. However, even though these systems allowed tight regula-tion of some mycobacterial genes, they only showed limited or no phenotypic regulation for others. By adapting their codon usage to that of the Mycobacterium tuberculosis genome, we created tetR genes that mediate up to ~50-fold better repression of reporter gene activities in Mycobacterium smegmatis and Mycobacterium bovis BCG. In addition to these repressors, for which Anhydrotetracycline (atc) functions as an inducer of gene expression, we used codon-usage adaption and structure-based design to develop improved reverse TetRs, for which atc functions as a corepressor. The previously described reverse repressor TetR only functioned when expressed from a strong promoter on a multicopy plasmid. The new reverse TetRs silence target genes more efficiently and allowed complete phenotypic silenc-ing of M. smegmatis secA1 with chromosomally integrated tetR genes
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controlling gene expression in mycobacteria with Anhydrotetracycline and tet repressor
Nucleic Acids Research, 2005Co-Authors: Sabine Ehrt, Xinzheng V Guo, Christopher M Hickey, Marvin Ryou, Mercedes Monteleone, Lee W Riley, Dirk SchnappingerAbstract:Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using Anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.
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doi:10.1093/nar/gni013 Controlling gene expression in mycobacteria with Anhydrotetracycline and Tet repressor
2004Co-Authors: Sabine Ehrt, Xinzheng V Guo, Christopher M Hickey, Marvin Ryou, Mercedes Monteleone, Lee W RileyAbstract:Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slowgrowing mycobacteria, including Mycobacterium tuberculosis, using Anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome
Rolf Lutz - One of the best experts on this subject based on the ideXlab platform.
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Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR/O, the TetR/O and AraC/I1-I2 Regulatory Elements
Nucleic Acids Research, 1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.
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independent and tight regulation of transcriptional units in escherichia coli via the lacr o the tetr o and arac i1 i2 regulatory elements
Nucleic Acids Research, 1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.
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Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements
1997Co-Authors: Rolf Lutz, Hermann BujardAbstract:Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with Anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of P lac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd g..
Francois Wasels - One of the best experts on this subject based on the ideXlab platform.
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a two plasmid inducible crispr cas9 genome editing tool for clostridium acetobutylicum
Journal of Microbiological Methods, 2017Co-Authors: Francois Wasels, Jennifer Jeanmarie, Florent Collas, Ana M Lopezcontreras, Nicolas Lopes FerreiraAbstract:CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an Anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.
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A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum
Journal of Microbiological Methods, 2017Co-Authors: Francois Wasels, Florent Collas, Jennifer Jean-marie, Ana M. López-contreras, Nicolas Lopes FerreiraAbstract:CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an Anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6 kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.