Team:WashU StLouis/Demonstrate

Validating a Part: Ribitol Transport BBa_K2663000

In order to test the efficacy of our ribitol transporter (RT) gene, we designed an experiment where we incubated both E. coli containing the transformed plasmid and wild-type E. coli DH5a in M9 minimal media with 0.2% by mass ribitol for less than 24 hours. We took samples and harvested the intracellular fluid at 2 time points. Concentrations of ribitol in the intracellular fluid were measured using Gas Chromatography-Mass Spectroscopy (GC-MS) with an organic solvent. The measured samples of both wild type and engineered E. coli were then compared, and basic statistical analysis was done to show that the average concentration of intracellular ribitol (per unit biomass) was more than seven times the baseline ribitol concentration of the wild-type cells.

Given that we had only incubated the cells for less than 24 hours in the ribitol-enriched M9 media, this is a promising result for our transporter. In future experimentation, we hope to do longer incubation periods with varying concentrations of ribitol. Literature (Pfyffer and Rast 1979) has proven that there are relatively high (20 mg per gram dry fungal mass) concentrations of ribitol in the Pgt fungus, so even a low amount of transport into the cell would be sufficient for our purposes.