Difference between revisions of "Team:BroadRun-NorthernVA/Results"
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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. | 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. | ||
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Revision as of 14:56, 18 September 2015
{{BroadRun-NorthernVA}}
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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 and 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.
After two hours, 200µl of iodine was added to the third well of starch and yeast solution and 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. 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 pipe. 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 pipe.
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.
Here is an image comparing all three wells side by side.
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