Following the insertion of genetic constructs into vectors and transformation into yeast, the genetically engineered yeast cells were tested for starch degradation in known starch solutions. Three yeast strains were successfully engineered, each producing a different amylase enzyme. The yeast strains were then tested in solutions of starch to determine if the amylase enzyme was being produced.
As seen in our results below, we proved that the yeast were indeed producing functional amylase enzymes. This confirms that our Biobrick devices work as expected.
Testing in Known Starch solutions
To characterize starch degradation in a controlled environment, the yeast strains were tested in starch solutions of a known concentration. Four yeast strains were tested; 3 genetically modified (labeled Construct 1, Construct 2, and Construct 3 for simplicity) and 1 wild type strain, used as a control.
Short Term Testing
In the first test, 4 yeast strains were added to 0.5% starch solution in a 1:1 ratio. Undiluted samples were measured every hour for 6 hours, using iodine to cause a color change.The intensity of the color change correlates to the concentration of starch.
A spectrophotometer was used to quantify the intensity of color change. A spectrophotometer sends specific wavelengths of light through a sample and measures how much of the light was absorbed. If there is a high concentration of a substance in a solution, more light will be absorbed. Thus, higher spectrophotometric absorbance values correlate to higher quantities of starch. A higher starch concentration results in a darker blue color change, which causes more light to be absorbed, and thus a higher absorbance value.
Figure 1. Color change of starch solutions showing starch degradation at 5 and 6 hours. In the above images, a difference in color change is apparent. After 5 and 6 hours, the genetically modified yeast strains (C1, C2, C3) have largely degraded the starch; the low concentration of starch is indicated by the pale blue color. In contrast, the control, the wild type yeast (wt), has a darker blue color, indicating a higher concentration of starch.
Figure 2. Quantified starch degradation over 6 hours for three genetically modified strains and a control. The graph above shows the absorbance values for each yeast strain over time. The absorbance values correspond to the concentration of starch. An absorbance value of 1 corresponds to approximately 0.25% starch. All samples started with an absorbance value of ~1.
All three of the genetically modified strains degraded nearly all of the starch within 6 hours, indicated by the change in absorbance from a value of 1 to nearly 0. These results prove that the yeast are indeed producing and secreting functional amylase enzyme. In contrast, the level of starch for the control remained nearly the same throughout the 6 hours. The slight decrease seen in the control can be attributed to the very small amount of amylase enzyme that wild type yeast naturally produce.
Long Term Starch Degradation Testing
With confirmation that the yeast were producing amylase enzymes, the next test was to determine the rate of degradation over a longer period of time.
The 4 yeast strains were added in a 1:8 ratio of 3% starch, with a total volume of 250mL. By decreasing the yeast to starch ratio, and increasing the concentration of starch, it was ensured that there would be sufficient starch for the yeast to break down over a period of several days. Samples were diluted with water in a 1:8 ratio, iodine added to cause a color change, then absorbance was measured in the spectrophotometer every 24 hours, for 72 hours.
Figure 3. Color change of starch solutions 48 hours after yeast was added.In the above image, a difference in color change is apparent 48 hours after the addition of the yeast. The 3 genetically modified yeast strains (C1, C2, C3) have degraded a large portion of the starch. This is indicated by the pale, translucent blue seen in the three tubes on the right. The control, wildtype yeast, has a far darker, opaque blue color, indicating a much higher concentration of starch.
Figure 4. Starch degradation quantified over 72 hours using spectrophotometric absorbance values. Here, an absorbance value of ~1.1 corresponds to ~2% starch.
Within 72 hours all three of the genetically modified strains degraded a significant amount of the starch, indicated by the decrease in absorbance from 1.9 to nearly zero. Construct 3 appears to be the most efficient in degrading starch in the long term. The control, however, had almost no change in starch concentration. The slight decrease seen for the control can be attributed to the small amount of amylases that wildtype yeast produce.
For the genetically modified strains, the steep decline in absorbance after 24 hours, followed by a less steep decrease at 48 and 72 hours, is likely due to a high starting amount of amylase in the solution. Before the yeast were added to the starch solution, while the yeast were growing in liquid culture they were producing and secreting amylase into the liquid media. When this amylase rich yeast culture was added to the starch solution, the excess of amylase enzyme rapidly degraded starch. As this initial quantity of excess enzyme was used up after ~24 hours, the remaining starch began to be broken down by the amylase enzyme the yeast produced after being added to the starch solution. The rate at which the yeast produce the enzyme is lower than the rate at which the excess starting amount of amylase can break down starch, thus there is a lower rate of degradation from 24 to 72 hours than from 0 to 24 hours.
