Successful Transformation and Plasmid Integration
We successfully transformed E. coli cells with our engineered plasmid containing the gene for ampicillin resistance. This was confirmed by the positive growth of all our transformed cultures on selective media, while the negative controls (non-transformed cells) further strengthened the case for the transformation working, as they displayed no growth. This confirmed the integration of our plasmid into the cells and the expression of the desired gene, allowing for further testing of the oxalate-degrading enzyme, oxalate oxidase.
Oxalate Degradation Experiment
After confirming the successful transformation of the cells, we proceeded with testing the enzyme’s ability to degrade oxalate, the key component in kidney stone formation.
The transformed cells were lysed to release the oxalate oxidase enzyme. Despite facing challenges with some of the lysis solutions due to expired reagents and potential protease contamination, we were able to obtain valid samples that demonstrated measurable oxalate degradation, as our assay displayed a clear degradation of oxalate. It was even visually evident which of our test had worked, as it was clearly more coloured than the ones that did not.
Figure 1: Growth of E. coli on agar plates with and without ampicillin (AMP)
This figure shows the results of a transformation experiment using E. coli on agar plates with and without ampicillin.
- Plate 1 (Top left): Negative control with AMP but without cells, showing no growth.
- Plate 2 (Top center): Negative control with non-transformed E. coli cells on AMP, showing no growth as expected since the cells lack ampicillin resistance.
- Plate 3 (Top right): Transformed E. coli cells with AMP, showing significant growth, confirming successful transformation and acquisition of ampicillin resistance.
- Plate 4 (Bottom left): Another replicate of transformed E. coli cells with AMP, again showing successful growth, further validating the transformation results.
- Plate 5 (Bottom center): Positive control with normal E. coli cells grown on LB media without AMP, showing expected growth in the absence of selective pressure.
- Plate 6 (Bottom right): Another replicate of the positive control with LB media, confirming normal cell growth without the presence of ampicillin.
The plates clearly demonstrate the selection pressure imposed by ampicillin, where only transformed cells with the resistance gene were able to grow.
Kinetic Data: Degradation Over Time
To quantify the enzyme’s activity, we tracked the oxalate degradation over three different time points: 1.5 hours, 3.5 hours, and 8 hours. Each time point demonstrated an increasing reduction in oxalate concentration, which correlates with the enzyme’s active degradation of the substrate.
The data followed an expected kinetic pattern typical of enzyme-substrate reactions:
- Initial Phase: In the early stages (1.5 and 3.5 hours), the enzyme exhibited a rapid reduction in oxalate concentration, indicative of a high substrate availability.
- Later Phase: By 9 hours, the reaction rate slowed, resulting in a plateau-like effect. This decrease in reaction rate is characteristic of enzymatic reactions, where substrate concentration limits the reaction speed as the enzyme becomes saturated or the substrate is depleted.
The figure below shows the oxalate degradation curve, reflecting the enzyme’s kinetics. The curve follows a non-linear trend, which aligns with the theoretical model of enzyme activity as the substrate concentration decreases over time.
Figure 2: Oxalate degradation over time by the enzyme oxalate oxidase. The curve illustrates the enzyme's kinetic behavior, with a rapid decline in oxalate concentration in the early stages and a slower reaction rate as the substrate is depleted
Conclusion
The experimental results strongly suggest that oxalate was successfully degraded by the enzyme oxalate oxidase produced by the genetically modified E. coli. The enzyme's behavior closely followed the predicted kinetics of enzyme-substrate reactions, with rapid degradation in the early phases and a plateau as the substrate concentration diminished. These results provide further validation that genetically modified bacteria could be effective in reducing oxalate levels, potentially serving as a therapeutic approach for preventing kidney stones.
