Team:BroadRun-NorthernVA/Results

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Project Results

We had successfully genetically engineered yeast cells that could produce and secrete the alpha amylase enzyme. Now we had to test it. First, we tested the modified strain of yeast in a solution of soluble starch combined with distilled water.

We started with a stock solution of 1% soluble starch solution and did 7 two fold dilutions, ending with .0078% starch concentration. This was completed in a 48 well plate. 200µl of diluted iodine was added to each of the wells. The iodine reacted with the starch in the water and turned a blue color. The starch concentration directly correlated with the color intensity. This first row of the well plate was used as a reference to discern the concentrations of starch left after the yeast cells had been left for a few hours.

Next we had to prove proof of concept, confirm that the yeast cells were indeed producing a functional form of the alpha amylase enzyme.

First, we tested the modified strain of yeast in a solution of soluble starch combined with distilled water. We started with 200µl of a .0625% concentration of starch. This was pipetted into three wells. 200µl of our yeast cell liquid cultures were added to the three wells, and left for one hour and two hours. Then iodine was added. The iodine reacts with the starch and changes color, the color intensity is directly correlated to the starch concentration. This would enable us to see how much starch had been broken down by the yeast cells in a given period of time.

The first well, which was treated with 200µl of iodine immediately after adding the cells, is a dark blue color. A large dark, almost black, spot of starch molecules that didn’t dissolve in the water and settled to the bottom of the well is clearly discernible, showing there is still a significant amount of starch in the water.



To the second well, 200µl of iodine was added to the starch and yeast solution after one hour. Then the well was photographed. The second well, which had the amylase-producing yeast cells added to it for one hour, is still blue, but a much lighter shade. Also the dark spot of starch molecules at the bottom of the well has decreased slightly in size, showing that some of those starch molecules have been broken down.



To the third well, 200µl of iodine was added after two hours to the starch and yeast solution. Then the well was photographed. The third well, which had the amylase-producing yeast cells added to it for two hours, has almost clear water, with only a very light tint of blue. This means the water has a much lower concentration of dissolved starch than before. The dark spot of undissolved starch molecules at the bottom of the well has significantly decreased in size, by more than half.



Here is an image comparing all three wells side by side.

These results show that while in the solution, the yeast cells produced and secreted the alpha amylase enzyme out of the cell. The amylase enzyme broke down and hydrolyzed the starch molecules in the water solution into monosaccharides, greatly reducing the amount of starch in the water within just a few hours.

Testing in Industrial Water Samples

Once we had established that our yeast cells were producing and secreting the amylase, we wanted to test if our cells could function as effectively in the industrial water system of Armstrong’s factory. Armstrong sent us two water samples from their factory, one from the aeration basin, and one from a tray used to hold excess water. Using our known concentrations of starch and iodine from the two fold dilutions, we determined that the Armstrong water sample contained about .006% starch, a very small amount.

We repeated the same process as before, adding 200µl of the water samples followed by 200µl of liquid yeast cultures in the three wells. The upper well is the water sample from the aeration basin and the lower well is the water sample from the tray.



The first two wells was photographed immediately after the addition of the yeast cells and iodine. Since the level of starch is so low, a clear color change cannot be seen in the water itself. The dark tinted spot at the bottom of the wells are the starch molecules that have settled down to the bottom of the well. A color change can be seen there, the starch has reacted with the iodine.

The second set of wells had 200µl of iodine added after one hour, and was then photographed. The dark spot, the settled starch molecules at the bottom of the wells, has decreased in size significantly, showing that the starch concentration has decreased as well.



The third set of wells had 200µl of iodine added after two hours, and was then photographed. The spot of settled starch molecules at the bottom of the wells is no longer visible, meaning the starch has been broken down by the amylase produced by the yeast cells.



Below is an image comparing all three wells side by side. The upper row of wells is the water samples from the aeration basin, and the lower row is the water samples from the tray.



These results have two major significances in terms of both our project and Armstrong’s industrial water issue. First, our genetically modified yeast cells are able to survive and thrive in the industrial waste water conditions. If they were to be added to Armstrong’s factory, the cells would still be able to reproduce and continue produce the amylase enzyme. This makes our solution a long term and sustainable, the yeast cell supply would not have to be periodically added in again. Second, our yeast cells are able to produce and secrete the amylase enzymes, as proven in both of the tests (soluble starch and industrial water sample). The amylase enzyme very effectively and quickly broke down the starch molecules in the water. In the industrial water sample, nearly all of the starch molecules were broken down by the amylase enzyme within two hours.



Future Work Our current amylase expression system has three major components, each of which can be improved upon as we work further on this project.
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• It is possible to strengthen the 'promotor so that the yeast produces more enzyme. However, this would probably produce a greater metabolic burden on the yeast. Therefore, we would probably also need to choose a promoter that turns protein expression on only in the presence of an external stimulus. This stimulus could be something associated with the buildup of starch in the environment or if could be a signal that we deliberately introduce to tell the yeast to start making the amylase.
• The secretion signal is also important to our design. It allows the yeast to secrete the amylase directly into the environment so that it can break down the starch. If we can improve the efficiency of the secretion signal, it will increase the amount of enzyme in the environment.
• Lastly, there are plenty of opportunities for us to improve upon the amylase itself. For example, we may be able to shorten the amylase protein to increase its expression efficiency. We may also be able to find an amylase which is more enzymatically active against starch in question.

We also want to modify the system based on how we intend to use the yeast. For example we could put in a "kill switch" to ensure that the yeast doesn't escape into the environment. We have also considered adding a protein tag to the enzyme so that the secreted amylase can be bound to a solid surface, preventing it from washing out of the pipe.