Quantifying Gene Expression in Pneumococcus

A modified protocol for qPCR was performed based on previously published methods (43 (link)). Spn strains were streaked and grown through secondary culture as above, grown in triplicate. One culture remained untreated, one was treated with 1/10th MIC, and the final culture was treated with 192 μg/mL erythromycin. Cultures were grown for 1 h before the bacteria were pelleted at 4K rpm for 5 min and frozen at −80°C. Genomic DNA was isolated from the pellets using the Zymo Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research) according to the manufacturer’s protocol. Genomic DNA concentration was determined using a NanoDrop 8000 spectrophotometer. The samples were diluted to 15 ng/μL, and qPCR was performed with standard curves using 10-fold serial dilutions of untreated genomic DNA. Gene copy numbers were determined relative to the fab(K) control using the ΔCt method.