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Supplemental Material for Neve, et al., 2019

posted on 2019-11-11, 14:20 authored by Isaiah A. A. Neve, Jessica N. Sowa, Chih-Chun Lin, Priya Sivaramakrishnan, Christophe Herman, Youqiong Ye, Leng Han, Meng C. Wang

Metabolomics analysis

E. coli metabolite profiling was performed at Metabolon, Inc (Durham, NC) with 5 biological replicates of each test bacterial strain (MG1655, OP50, HB101). E. coli colonies were inoculated in LB, grown overnight, and then plated on Nematode Growth Medium (NGM) Agar plates to form lawns for three days at 20°C. Lawns were then scraped off of the surface of the agar and collected in distilled deionized water. Samples were then washed 3 times. Each sample contained approximately 0.1mg of pooled bacterial pellet which was then flash frozen using liquid nitrogen. Metabolomics profiling was performed in the method described previously (Evans et al. 2009). Statistical analysis was performed using Welch’s two-sample t-test to identify compounds that differed significantly between bacterial strains.

Methodology for performing Reproductive Lifespans with Betaine supplementation

Reproductive lifespans (RLS) were performed at 20°C on animals that were developmentally synchronized using a variation of the “egg prep” methodology described in (Porta-de-la-Riva et al. 2012). Well-fed animals were raised for two generations on standard NGM plates seeded with OP50. The third generation of animals were plated on antibiotic free NGM plates seeded with the test bacteria, and if necessary, an inducible agent (IPTG) or supplement (Betaine at 20mM during development, and 100mM after L4, based upon plate volume). Upon development to the L4 stage, worms were singled to individual plates containing their specified food source. The animals were then transferred to fresh plates every day at the same time until reproduction cessation, determined as the point when no progeny were produced for three consecutive days. Two days after the individual worms were transferred, plates were checked, and double checked for progeny. Animals that bag or die were noted as censors. Reproductive span curves were calculated using Kaplan-Meier survival analysis and compared using the log-rank test. At least two independent trials were conducted.

PCR of stated loci was conducted with primers listed


Article title

Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans

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