p450 gene abundance measurements in the COAST mesocosm study

A mesocosm experiment was carried out in and October of 2015 (Mesocosm#2) using coastal water collected near Galveston Bay, Texas. Four mesocosm treatments were prepared in triplicate for this study: (1) Control (seawater only); (2) WAF (oil + seawater); (3) Chemically-enhanced WAF (CE-WAF; oil + Corexit + seawater); (4) Diluted CE-WAF (1:10 dilution). Initial mesocosm volumes were 79L. Prior to experiment initiation, phytoplankton were collected from coastal Galveston, TX waters and added to each tank to stimulate interactions between phytoplankton, oil, and Corexit. Starting at time-point zero and every 12 hours thereafter, water samples were collected from each mesocosm in clean, opaque, Nalgene bottles. For each timepoint, 150mL of water was filtered onto 47mm, 0.2um filters and DNA was extracted using MP Biomedical FastDNA spin kits. Primers P450fw1 (5’-GTSGGCGGCAACGACACSAC-3’) and P450rv3 (5’-GCASCGGTGGATGCCGAAGCCRAA-3’) were used to obtain PCR products for alkane monooxygenase P450 (van Beilen et al., 2006). PCR products were ligated into competent E. coli cells and cloned using the TOPO TA Cloning Kit (Life technologies; Carlsbad, CA). Plasmid extractions were performed using Qiagen Mini-prep Kits. Sequencing of 7 random clones verified the success of the cloning reaction and exact size and sequence of the targeted PCR products were confirmed with BlastX. A mixture of five P450 clone sequences were used to create a standard for qPCR, four Alphaproteobacteria and one Gammaproteobacteria. qPCR was carried then out for on all DNA samples in triplicate.