Fluorescence Resonance Energy Transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster() DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4+/-3.0x10(6) and 5.4+/-3.0x10(2)CFUml(-1) cell suspension. The detection limit was about 540CFUml(-1), which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4+/-3.0x10(4)CFUg(-1) dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.

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Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Phrommanich

Suanjit

Upatham

et al. 2009

Microbiological Research

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Establishment of DsRed.T3_S4T as an improved autofluorescent marker for microbial ecology applications

Dandie

Larrainzar

Mark

et al. 2005

Environmental Microbiology

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SummaryAutofluorescent proteins (AFPs), such as green fluorescent protein (GFP) and DsRed, are valuable tools for studying plant-microbe interactions. Nevertheless, because of some limitations, efforts are ongoing to generate improved AFP variants. Several groups have generated variants of GFP with altered spectral characteristics, and faster maturing and brighter variants of DsRed. In this study we used plasmid and chromosomal constructs to test the efficacy of a new variant of DsRed, DsRed.T3_S4T, in Pseudomonas fluorescens F113rif. In addition, we compared the ecological fitness of strains carrying chromosomal copies of EGFP , DsRed or DsRed.T3_S4T . Strains expressing DsRed.T3_S4T fluoresced significantly brighter than strains expressing DsRed . Furthermore, it was found that although all strains grew equally well in vitro , only strains carrying DsRed.T3_S4T functioned as well as wild type in a competitive rhizosphere colonization assay. In particular, it was observed that DsRed.T3_S4T is an improved marker over DsRed for microbial ecology studies in this strain.

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Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment

Andõ

Suzuki

Shima

2005

Appl Environ Microbiol

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Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (ATH1) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study, ATH1 disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains ATH1 loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of ATH1 by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.

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Fluorescence Resonance Energy Transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil

Cited by 5 publications

References 27 publications

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Establishment of DsRed.T3_S4T as an improved autofluorescent marker for microbial ecology applications

Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment

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