RESUMEN
By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.
Asunto(s)
Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Xilosidasas/metabolismo , Biotecnología/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Calor , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Xilosidasas/genéticaRESUMEN
Fluorescent proteins (FPs), such as green fluorescent protein (GFP) and its variants, are well-developed visible markers for analyzing bioprocesses. Accurate measurement of fluorescence emitted from FPs in whole cells is complicated by the inner filter effect (IFE), which is caused by intracellular light absorption and scattering by cell particles. The IFE causes nonlinearity between fluorescence intensity and fluorophore concentrations in FP-harboring cells and can significantly influence the accuracy of FP-based analysis, especially at high cell densities. A mathematical model based on detection of fluorescence intensity using a fluorescence spectrophotometer was developed to provide a simple correction for the IFE in fluorescence intensity detection in high-density cultures. The parameters of this model were determined in three different FP-harboring bacterial strains to give the "real fluorescence" intensity without the IFE. Using these parameters, accurate analysis of FP-labeled Escherichia coli at high cell density in pure culture and in mixed cultures with fluorescent and nonfluorescent strains was easily and successfully achieved.
Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Espectrometría de Fluorescencia/métodos , Escherichia coli , Filtración , Proteínas Fluorescentes Verdes/genética , Modelos Teóricos , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
OBJECTIVE: To investigate the optimal operative approach for the complicated anal fistula. METHODS: One hundred and ninety-two cases with complicated anal fistula were randomly divided into minimally invasive operation group (through spatium intermuscular of anal sphincter) and fistula resection group. The operation time, bleeding time during and after operation, pain lasting time, healing time of incision, area of anal scar, anal malformation and function and post operative recurrence were observed and compared between the two groups. RESULTS: Compared to those of fistula resection group, the operative time was (36.5+/- 15.3)min, bleeding time during and after operation (2.0+/- 0.5)d, postoperative pain lasting time (1.5+/- 0.5)d, healing time of incision (18.5+/- 5.5)d in minimally invasive operation group. All were shortened (P< 0.05), and the incidence of anal malformation (5.2%, P< 0.01) and partial anal incontinence (2.1%, P< 0.01) was lower. There was no significant difference in postoperative recurrence between the two groups. CONCLUSIONS: The minimally invasive operation through spatium in termuscular of anal sphincter is superior to fistula resection on the management of complicated fistula.
Asunto(s)
Canal Anal/cirugía , Procedimientos Quirúrgicos Mínimamente Invasivos , Fístula Rectal/cirugía , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Green fluorescent protein (GFP) can be utilized in analysis of the characteristics and distribution of a targeted strain in microbial communities. This study is the first step to establish a dynamic yeast monitoring technique in a wastewater treatment system using yeast by constructing a fluorescent yeast containing gfp gene. The gfp gene was inserted into pACT1-URA3, a powerful plasmid for introducing a foreign gene into Candida boidinii, and then transformed into E. coli JM109. The gfp gene was expressed, though not very highly. The results of the electrophoresis and polymerase chain reaction suggested that the newly constructed plasmid containing gfp gene might not exist in free form in the cells, but in some special way such as interaction with the chromosome.