Part:BBa_K5422007:Experience

Objective

The objective of our work was to measure the effect of a catabolic competitor more specifically glucose on the activity of the Pxyl promoter. This experiment represents our contribution to the characterization of the BBa K733018 part.

Method

The induction of the Pxyl promoter was investigated using the W3110 strain containing the plasmid pOK12 with the pXyla-GFP. Cultures were prepared from two 2YT growth media, with or without the addition of ampicillin at 100 µg/mL. The optical densities (OD) measured at 595 nm for the initial cultures were 7.6 for W3110 and 7.4 for the XXb6 strain.

Cultures were diluted tenfold to inoculate 2 mL of media, adding 27 µL of the cell dilution (ODi = 0.01). The incubation was carried out at 37°C with shaking at 200 rpm. Measurements of OD at 595 nm and fluorescence were taken in the stationary phase to assess the impact of different xylose concentrations on the expression of the GFP reporter gene.

Induction conditions varied with different xylose percentages (0%, 0.015%, 0.05%, 0.1%, 0.15%, 0.5%, and 1%), combined with the presence or absence of glucose. Volumes of xylose at 10%, 20%, and 50% were adjusted accordingly for each condition. For instance, for 0.5% xylose, 15 µL of 20% xylose solution was added, while for 1%, 40 µL of 50% xylose was used.

Characterization Procedure

Fluorescence measurements were performed using a Tecan reader at a single time point in the stationary phase. Each data point represents the average of two biological replicates, as the third replicate was not comparable.

Results

Analysis of Pxyl promoter induction by xylose revealed significant differences between conditions with and without glucose. In the absence of glucose, increasing xylose concentrations resulted in elevated fluorescence levels. The estimated KM was 33.2 ± 18.5 mM, indicating a low affinity of the promoter for xylose.

In contrast, in the presence of glucose, fluorescence was significantly reduced at xylose concentrations below 40 mM, demonstrating an inhibitory effect of glucose on promoter induction.

This phenomenon aligns with the principle of catabolite repression, where glucose, as the preferred carbon source, suppresses the utilization of alternative sugars such as xylose. At higher xylose concentrations, the inhibitory effect of glucose appeared to diminish, allowing fluorescence to approach levels observed without glucose.

Discussion

These findings highlight the importance of glucose concentration in regulating xylose-induced expression. Further investigations may be needed to explore the complex interactions between glucose and xylose (or other monosaccharide) in promoting the Pxyl activity, as well as to determine the implications of the estimated KM for optimizing induction strategies in future applications.