Glucose-xylose growth experiments

To investigate the effectiveness of our xylose-utilizing module, we transformed the plasmid containing the native XylR gene and that containing the mutated XylR (XylR*) into E. coli BL21*. Since XylR acts as a co-activator of the xylose operon, we hypothesized that overexpressing native XylR would also help to enhance xylose utilization. Also, the effect would be more pronounced when XylR* was expressed, as it has significantly higher binding affinity. BL21*, BL21*-XylR, and BL21*-XylR* were grown in 0.2% glucose, 0.2% xylose, as well as a mixture of 0.1% glucose and 0.1% xylose. Growth, as an indicator of sugar substrate utilization, was measured via absorbance at 600nm over 8-hours in a microplate reader.

To verify the expression of XylR and XylR*, SDS-PAGE was conducted for samples taken after induction. Thick bands at approximately 45kDa were observed for induced cells, with no corresponding band for the WT and uninduced samples, showing that XylR and XylR* are overexpressed respectively (Figure 1).

Our growth experiments displayed rather interesting results. BL21*-XylR* had noticeably the highest growth in all three conditions, while BL21*-XylR displayed considerably little or even no growth (Figure 2a, b, and c). Most importantly, overexpressing XylR* seemed to increase xylose utilization, where the growth of Bl21*-XylR* is significantly higher than the wild-type in xylose (Figure 2e), as well as in a mixture of glucose and xylose (Figure 2f). Our xylose-utilization module likely works!

Surprisingly, XylR* also appeared to enhance growth in glucose (Figure 1a). We expected it to have similar growth as the wild-type instead. Hence, it is difficult to ascertain whether the augmented growth in xylose is a result of improved utilization or other mechanisms, which is possible due to XylR’s role as a metabolic regulatory factor.

Nonetheless, BL21*-XylR* exhibited comparable growth rates in glucose and the mixture of glucose and xylose, suggesting co-utilization of sugar substrates occured (Figure 1f). In contrast, this was not observed in the wild-type, where having a mixture of sugars resulted in lower growth rates (Figure 1d). This is significant as we are one step closer to our vision of utilizing lignocellulosic waste as feedstock, which requires simultaneous utilization of glucose and xylose.

As for BL21*-XylR, there was little to no growth observed regardless of the sugar substrates (Figure 2e). Not only does XylR overexpression fail to improve xylose utilization as hypothesized, there appears to be a growth inhibitory effect. It is possible that other metabolic processes were compromised.

All in all, the XylR*-overexpressing xylose utilizing module is likely to be functioning as intended, but further tests would be necessary. To obtain a more definitive indication of glucose and xylose utilization, HPLC analysis could be carried out on the medium. If xylose utilization was not the contributing factor to enhanced growth, further tests could be conducted to elucidate the mechanism.

On the other hand, if XylR* overexpression does increase xylose utilization as expected, there are plenty of possibilities to be explored! To further demonstrate the applicability of our xylose utilizing module, crude lignocellulosic waste extracts can be fed as well. We can co-transform our current module with our biosynthesis plasmids, demonstrating the production of naringenin or even luteolin from xylose and bringing us closer to our final system. By varying inducer concentrations, the optimal level of XylR* expression can be determined. As such, other strategies could be employed to attain the level of expression without using chemical inducers - one of the key objectives of Coup Dy'état. This includes constitutive or light-regulated expression, or even metabolic engineering. More excitingly, a xylose-based nutrient-sensing module could conceivably be developed, allowing for dynamic regulation via light induction!

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