Part:BBa_K5422009:Experience

Objective

The goal of this experiment was to characterize the functionality of the PxylA-RBS-control receptor construct by evaluating its protein expression levels at different xylose concentrations.

Method

We performed a western blot to assess the production of the control receptor protein. The expression of the PxylA-RBS-control receptor was induced by varying concentrations of xylose: 1%, 2%, 3%, 4%, 5%, and 10%, alongside a non-induced control (0% xylose). A second experiment was conducted using lower concentrations of xylose: 0%, 0.05%, 0.2%, 0.4%, 0.6%, and 0.8%.

Results

The results indicate that the PxylA promoter reaches a saturation point at xylose concentrations above 1%, beyond which additional xylose does not induce further protein expression. This was evident from the consistent protein levels observed across the 1% to 10% xylose concentrations, with no increase in production despite rising inducer levels. The presence of the control receptor protein at the expected size of approximately 35.5 kDa was confirmed, but the lack of increased production with higher xylose concentrations suggested that these levels may have saturated the inducible promoter. Once this saturation threshold was reached, adding more xylose did not lead to further protein expression, indicating a limit to the promoter’s inducibility.

In contrast, the second experiment using lower concentrations of xylose (ranging from 0.05% to 0.8%) demonstrated a more gradual and proportional increase in control receptor protein production. The results of this second western blot validated our hypothesis, confirming that xylose concentrations above 1% indeed saturate the promoter, preventing additional induction of protein expression. Thus, lower xylose concentrations proved more effective for finely tuning protein expression levels in this system.

Discussion

This experiment highlights the importance of optimizing inducer concentrations to avoid promoter saturation. The data suggest that concentrations above 1% xylose are unnecessary for achieving optimal protein expression with the PxylA promoter. Future experiments could explore the effects of other carbon sources, such as glucose, to further investigate potential catabolite repression effects on the control receptor's expression.