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In JoVE (1)
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Articles by Erin B. Fichot in JoVE
JoVE General
Extração de DNA de alto peso molecular de Esteiras Microbianas
Nós fornecemos um protocolo melhorado para a extração de DNA de alto peso molecular de tapetes microbianos hipersalinos. Células microbianas são separados da matriz mat antes da extração e purificação DNA. Isso aumenta a concentração, qualidade e tamanho do DNA. O protocolo pode ser usado para outras amostras refratário.
Other articles by Erin B. Fichot on PubMed
Extraction of High Molecular Weight DNA from Microbial Mats
Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields >2 µg HMW DNA (>48 kb) per gram of mat sample, with A260:280 ratios >1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness.
Characterization and Quantitation of a Novel β-lactamase Gene Found in a Wastewater Treatment Facility and the Surrounding Coastal Ecosystem
Wastewater treatment plants (WWTPs) are engineered structures that collect, concentrate, and treat human waste, ultimately releasing treated wastewater into local environments. While WWTPs efficiently remove most biosolids, it has been shown that many antibiotics and antibiotic-resistant bacteria can survive the treatment process. To determine how WWTPs influence the concentration and dissemination of antibiotic-resistant genes into the environment, a functional metagenomic approach was used to identify a novel antibiotic resistance gene within a WWTP, and quantitative PCR (qPCR) was used to determine gene copy numbers within the facility and the local coastal ecosystem. From the WWTP metagenomic library, the fosmid insert contained in one highly resistant clone (MIC, ≈ 416 μg ml(-1) ampicillin) was sequenced and annotated, revealing 33 putative genes, including a 927-bp gene that is 42% identical to a functionally characterized β-lactamase from Staphylococcus aureus PC1. Isolation and subcloning of this gene, referred to as bla(M-1), conferred ampicillin resistance to its Escherichia coli host. When normalized to volume, qPCR showed increased concentrations of bla(M-1) during initial treatment stages but 2-fold-decreased concentrations during the final treatment stage. The concentration ng(-1) DNA increased throughout the WWTP process from influent to effluent, suggesting that bla(M-1) makes up a significant proportion of the overall genetic material being released into the coastal ecosystem. Average discharge was estimated to be 3.9 × 10(14) copies of the bla(M-1) gene released daily into this coastal ecosystem. Furthermore, the gene was observed in all sampled coastal water and sediment samples surrounding the facility. Our results suggest that WWTPs may be a pathway for the dissemination of novel antibiotic resistance genes into the environment.
