Abstract Title

Establishing a Method to Extract Plasmid DNA from Environmental Samples

Disciplines

Environmental Microbiology and Microbial Ecology

Abstract

Manure and wastewater treatment plant (WWTP) sludge used as fertilizer are two possible sources of antibiotic resistant bacteria introduced into the environment. Resistance genes can be transferred horizontally between bacteria by mobile genetic elements such as plasmids. Plasmids could be transferred between manure and/or WWTP sludge to soil. The overall goal of the project is to characterize the impact of such soil fertilization practices on antibiotic resistance dissemination by plasmids in soil. No reliable method for extracting plasmid DNA from environmental samples without cultivation is available. We hypothesize that by combining various existing protocols used for plasmid isolation from culture and total DNA extraction from environmental samples, we will be able to extract plasmid from environmental samples. Initial results showed the quantity and purity of plasmid DNA obtained as a limiting factor. The addition of a known plasmid prior to extraction to reduce the loss of indigenous plasmids throughout the extraction process, and the addition of a CsCl gradient to purify the plasmid fraction should overcome those issues and provide adequate purity for sequencing purposes.

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Establishing a Method to Extract Plasmid DNA from Environmental Samples

Manure and wastewater treatment plant (WWTP) sludge used as fertilizer are two possible sources of antibiotic resistant bacteria introduced into the environment. Resistance genes can be transferred horizontally between bacteria by mobile genetic elements such as plasmids. Plasmids could be transferred between manure and/or WWTP sludge to soil. The overall goal of the project is to characterize the impact of such soil fertilization practices on antibiotic resistance dissemination by plasmids in soil. No reliable method for extracting plasmid DNA from environmental samples without cultivation is available. We hypothesize that by combining various existing protocols used for plasmid isolation from culture and total DNA extraction from environmental samples, we will be able to extract plasmid from environmental samples. Initial results showed the quantity and purity of plasmid DNA obtained as a limiting factor. The addition of a known plasmid prior to extraction to reduce the loss of indigenous plasmids throughout the extraction process, and the addition of a CsCl gradient to purify the plasmid fraction should overcome those issues and provide adequate purity for sequencing purposes.