4th Annual WML Academic Symposium

A current focus of research is to identify novel enzymes within microflora communities that could be used to increase efficiency and lower operating cost of applications across multiple industries. The aim of this study was to construct a metagenomic library of the microflora community of intestinal bacteria from equine fecal samples in order to identify new novel enzymes. Fecal samples were obtained from horses fed a mixture of grain and perennial pasture and hay grasses. Several genomic DNA extraction and purification protocols were evaluated to include commercial kits and pretreatments prior to their use. A modified pretreatment method using bead beating with a lysis buffer containing high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA followed by a purification step using QIAamp columns resulted in the highest yield of DNA. This procedure was scaled up in order to obtain a large volume of purified DNA for downstream applications. Multiple electrophoresis techniques were evaluated for the best band resolution of large sized DNA fragments. Total genomic DNA was electrophoresed in 0.8% agarose with 1% polyvinylpyrrolidone (PVP) which resulted in the majority of the chromosomal DNA fragments being located between 23-33 kb. The DNA from this section of gel was extracted and used for the construction of a library that will be screened for novel enzymes. Currently, there is a need in the biofuel industry for cellulases which can aid in the production of ethanol. To identify clones expressing these enzymes, screening will be conducted on carboxymethylcellulose (CMC) agar plates.