RESUMO
An evaluation using paraffin oil based, Acheson 38 carbon paste electrodes modified with α-hydroxyisobutyric acid (HIBA) to preconcentrate f-elements cathodically is described. The modified paste was made by directly mixing solid HIBA into the carbon paste. A chemically reversible cyclic voltammogram for HIBA was observed on this modified carbon paste, which was found to be a non-Nerstian, single electron transfer process. Lanthanides (less promethium) were found to accumulate onto the electrode surface during a 30 s electrodeposition step at -0.4 V vs Ag/AgCl from 0.1 M LiCl. The elements were then stripped off into a 2% HNO(3) solution by an oxidative step at +0.8 V vs Ag/AgCl; quantitative removal from the electrode was confirmed by ICPMS. Ultratrace solutions with initial concentrations down to 5 parts per quadrillion (ppq) were preconcentrated in 5 min above our instrumental limit of detection (LOD) of around 1 ppt for lanthanides.
RESUMO
A multiple A-tract sequence has been identified in the promoter regions for the mxaF, pqqA, mxaW, mxbD and mxcQ genes involved in methanol oxidation in Methylobacterium extorquens AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter-xylE transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the mxaF and pqqA promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the mxbD and mxcQ promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to Plac of Escherichia coli, which is a weak constitutive promoter in M. extorquens AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in M. extorquens AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.
Assuntos
Proteínas de Bactérias/genética , Elementos Facilitadores Genéticos/genética , Regulação Bacteriana da Expressão Gênica , Metanol/metabolismo , Methylobacterium extorquens/metabolismo , Transcrição Gênica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Methylobacterium extorquens/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Regiões Promotoras GenéticasRESUMO
Using an expression vector containing p(mxaF'), a strong native promoter, expression of a model heterologous protein, haloalkane dehalogenase, from Xanthobacter autotrophicus GJ10 was achieved in the methylotrophic bacterium, Methylobacterium extorquens AM1. Although expression using the wild-type strain was <5% of total cell protein, expression at a level of 10% of the total cell protein was achieved in a mutant unable to synthesize poly-beta-hydroxybutyrate granules. Two other tested heterologous proteins, catechol dioxygenase and green fluorescent protein, were expressed at moderate levels in both wild-type and the PHB-negative strain. These results suggest that the M. extorquens PHB-negative strain is a possible platform for overexpression of heterologous proteins with labeled or unlabeled methanol as a starting material.
