4.5. Lifespan Assays Using Resveratrol or Novel Polyketides

Wild-type C. elegans N2 strain and eat-2 mutant strain were used in lifespan assays. C. elegans were maintained according to Stiernagle (2006) with minimal changes.³⁶ Resveratrol (50 μM, TCI Co.) or DMSO (as a control) was incorporated into 5 cm NGM agar plates by another lab member to ensure blinding of the assays. NGM plates used for lifespan assays also contained 90 μM FUdR (Sigma-Aldrich Co.) to ensure batch synchrony by preventing cell division and growth in newly hatched C. elegans.³⁷ Cultures of E. coli OP50 strain were grown in LB overnight at 25 °C and concentrated 10-fold by centrifugation and resuspension in a smaller volume of LB. OP50 was then spotted onto NGM agar plates as a food source for C. elegans and allowed to dry overnight at room temperature. C. elegans were cultured at 20 °C on the various NGM agar plates containing a lawn of E. coli OP50. The worms were examined daily and scored as dead if no response was observed when they were gently prodded with a platinum wire. C. elegans that were lost because of crawling on the walls of the agar plates were omitted from analysis. Survival analysis was conducted using the log rank test in the IBM SPSS Statistics 22 program.

For lifespan assays using novel polyketides, selected combinations of 0.5 mM starter acids and 1 mM extender acids together with 90 μM FUdR, 30 μg/mL kanamycin, 34 μg/mL chloramphenicol, 100 μg/mL ampicillin, and 0.1 mM IPTG were incorporated into NGM agar plates. The corresponding CoA ligases + 18xCHS E. coli constructs and constructs without 18xCHS were grown in LB with antibiotics overnight at 25 °C by another lab member to ensure blinding of the lifespan studies. Protein expression was induced by 0.1 mM IPTG for 3 h at 25 °C, and 0.5 mM starter acids and 1 mM extender acids were subsequently introduced to the culture. After incubation at 25 °C for another 3 h, the E. coli culture was concentrated 20-fold, seeded onto the respective NGM plates, and allowed to dry overnight at room temperature as the food source which can expose C. elegans to the biosynthesized polyketides concurrently. The lifespans of wild-type N2 C. elegans exposed to the CoA thioesters and novel polyketides were compared to the lifespans of N2 worms exposed to CoA thioesters only, using the log rank test.