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<p1>The two yeast plasmids used were pRS426 and pAG36. The pSB1C3 plasmid was also cut with restriction enzymes, so that the constructs could be inserted for submission to the Parts Registry. For what enzymes were used for each construct/plasmid, and exact quantities of reagents, please see the Notebook. All three constructs and 3 plasmids were digested correctly, verified with a gel. </p1> | <p1>The two yeast plasmids used were pRS426 and pAG36. The pSB1C3 plasmid was also cut with restriction enzymes, so that the constructs could be inserted for submission to the Parts Registry. For what enzymes were used for each construct/plasmid, and exact quantities of reagents, please see the Notebook. All three constructs and 3 plasmids were digested correctly, verified with a gel. </p1> | ||
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<p1>After confirmation that the restriction digest was successful, the DNA samples were purified. The constructs were PCR purified and the plasmids were gel extracted. The DNA was purified to remove the unwanted pieces of DNA leftover from the restriction digest. These unwanted pieces of DNA could prevent the construct from being properly inserted into the plasmid. </p1> | <p1>After confirmation that the restriction digest was successful, the DNA samples were purified. The constructs were PCR purified and the plasmids were gel extracted. The DNA was purified to remove the unwanted pieces of DNA leftover from the restriction digest. These unwanted pieces of DNA could prevent the construct from being properly inserted into the plasmid. </p1> | ||
− | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/1/19/T--BroadRun-Baltimore--Methods_Labwork1.jpg | + | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/1/19/T--BroadRun-Baltimore--Methods_Labwork1.jpg" style="width: 416px;height: 322px;"> |
<p1>The two methods of purification were used because of the different sizes of the pieces of DNA that were left after the restriction digest. The digested constructs had only very small pieces of DNA cut by the enzymes, these small pieces would be washed away during purification. The digested plasmids had large pieces of DNA several thousand base pairs long, thus these pieces had to first be separated by size with gel electrophoresis, then the desired piece of DNA was cut out and purified. This process prevents the plasmid from ligating back on itself, without the construct inside, during ligation. </p1> | <p1>The two methods of purification were used because of the different sizes of the pieces of DNA that were left after the restriction digest. The digested constructs had only very small pieces of DNA cut by the enzymes, these small pieces would be washed away during purification. The digested plasmids had large pieces of DNA several thousand base pairs long, thus these pieces had to first be separated by size with gel electrophoresis, then the desired piece of DNA was cut out and purified. This process prevents the plasmid from ligating back on itself, without the construct inside, during ligation. </p1> | ||
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<p1>The PCR reagents used were: forward and reverse primers, DNTPs, Taq polymerase enzyme, Taq buffer, and water. Colonies chosen with toothpicks were stirred into the mix of reagents, and then used to inoculate liquid cultures of LB broth. These liquid cultures were set aside in the incubator, to be used the next day for miniprepping the cells. To ensure that at least one colony from each plate was successful, 7-8 colonies per plate were selected for colony PCR. </p1> | <p1>The PCR reagents used were: forward and reverse primers, DNTPs, Taq polymerase enzyme, Taq buffer, and water. Colonies chosen with toothpicks were stirred into the mix of reagents, and then used to inoculate liquid cultures of LB broth. These liquid cultures were set aside in the incubator, to be used the next day for miniprepping the cells. To ensure that at least one colony from each plate was successful, 7-8 colonies per plate were selected for colony PCR. </p1> | ||
− | <p1>After running the samples through the thermocycler, and gel was run on the samples. The gel confirmed that the constructs 1 and 2 had been properly inserted into the pAG36 vector, while construct 3 had not.</p1> | + | <p1>After running the samples through the thermocycler, and gel was run on the samples. The gel confirmed that the constructs 1 and 2 had been properly inserted into the pAG36 vector, while construct 3 had not. All 3 constructs had been properly inserted into the pSB1C3 vector. </p1> |
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<h4>Miniprep </h4> | <h4>Miniprep </h4> | ||
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+ | <p1>Once it had been confirmed which colonies were successful, those colonies were mini-prepped, to extract the plasmids inside of the cell. The DNA samples with the pSB1c3 plasmid were submitted to the Registry, those with the yeast plasmids were transformed to yeast. </p1> | ||
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+ | <h4>Yeast Transformation </h4> | ||
+ | <p1>Following mini-prep, the next step was a yeast transformation. The yeast plasmids used the selectable marker gene URA3, the corresponding yeast strain was used. This means the URA3 yeast strain can’t produce uracil. The URA3 gene in the plasmid codes for uracil. Transformed cultures were plated onto uracil deficient media, so the yeast cells without the plasmid wouldn’t be able to produce uracil and wouldn’t form colonies.</p1> | ||
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+ | <p1>Yeast cultures were prepped by culturing in YPD broth, and using a hemacytometer to ensure the cells were at the right density, before pelleting the cells and resuspending them in a competent cell mixture. </p1> | ||
<IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/b/b0/T--BroadRun-Baltimore--Methods_YeastPrep.jpg" style="width: 731px;height: 549px;"> | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/b/b0/T--BroadRun-Baltimore--Methods_YeastPrep.jpg" style="width: 731px;height: 549px;"> | ||
+ | <p1><i> Image of yeast cultures being prepped for transformation.</i></p1> | ||
+ | <p1>Following the preparation, the cultures were transformed with the DNA samples using the lithium acetate and poly ethylene glycol method of transformation. (For more, see the Notebook.) The yeast transformation was successful for the three constructs (2 from 2016, one from 2015). </p1> | ||
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<IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/7/7e/Methods_yeastplates.JPG" style="width: 813px;height: 364px;"> | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/7/7e/Methods_yeastplates.JPG" style="width: 813px;height: 364px;"> | ||
− | + | <p1><i>Image of yeast agar plates with many colonies, taken 3 days after transformation.</i></p1> | |
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<hr> | <hr> | ||
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<h1> Testing</h1> | <h1> Testing</h1> | ||
<h2>Phase 1: Testing in Known Starch Concentrations</h2> | <h2>Phase 1: Testing in Known Starch Concentrations</h2> | ||
− | + | <p1>The first step of testing was to test in a controlled environment, where the yeast were tested in solutions of known starch concentration. Iodine was added to the starch solutions, which causes a color change. The intensity of this color change, which correlates to the amount of starch, was quantified using a spectrophotometer. The spectrophotometer sends a specific wavelength of light through a sample and measures how much light was absorbed by the sample, which correlates to how much of a substance is in the solution. 4 different yeast strains were tested: three genetically modified yeast (three different constructs) and wildtype yeast as a control. A calibration curve was created, which enabled us to correlate the absorbance values from the spectrophotometer with the amount of starch. </p1> | |
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<h4>Short Term Testing</h4> | <h4>Short Term Testing</h4> | ||
− | + | <p1>In this test, the 4 yeast strains were combined with 0.5% starch, in a 1:1 ratio, for 6 hours. Measurements of the amount of starch were taken every hour. Before actually adding starch to the yeast cultures, all cultures were checked using the spectrophotometer, to ensure all had the same cell density. In addition to iodine to the samples for the starch measurement, HCl was added. This was to stop the cell’s metabolic reactions, which affect the iodine and starch reaction. </p1> | |
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+ | <p1>Starch measurements were made using the following protocol: </p1> | ||
+ | <ul style="list-style-type:none"> | ||
+ | <li style="font-size:20px";>1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. | ||
+ | <li style="font-size:20px";>2. Add 0.5mL of this yeast/starch solution to a cuvette. | ||
+ | <li style="font-size:20px";>3. Add 0.3mL of 1M HCL to the cuvette. | ||
+ | <li style="font-size:20px";>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. | ||
+ | <li style="font-size:20px";>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. | ||
+ | <li style="font-size:20px";>6. Measure absorbance using the spectrophotometer. | ||
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<h4>Long Term Testing</h4> | <h4>Long Term Testing</h4> | ||
− | + | <p1>In this test, the 4 yeast strains were combined with 2% starch in a 1:8 ratio of yeast to starch. This lower ratio and higher concentration of starch was to ensure that the yeast would not break down all of the starch within just a few hours. This solution was aerated using an air pump and tubing, to keep oxygen levels in the sample high. </p1> | |
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+ | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/c/c0/Methods_StarchSetup.jpg" style="width: 768px;height: 576px;"> | ||
+ | <p1><i>Setup of long term starch degradation testing. The 4th flask, with wildtype culture, is not pictured.</i></p1> | ||
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+ | <p1>Measurements were made every 24 hours, for 72 hours, using the following protocol: </p1> | ||
+ | <ul style="list-style-type:none"> | ||
+ | <li2 style="font-size:20px"; >1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. | ||
+ | <li2 style="font-size:20px";>2. Add 0.5mL of this yeast/starch solution to a cuvette. | ||
+ | <li2 style="font-size:20px";>3. Add 3.5 mL of water and 0.3mL of 1M HCL to the cuvette. | ||
+ | <li2 style="font-size:20px";>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. | ||
+ | <li2 style="font-size:20px";>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. | ||
+ | <li2 style="font-size:20px";>6. Measure absorbance using the spectrophotometer. | ||
<h2>Phase 2: Testing in Industrial Water Samples</h2> | <h2>Phase 2: Testing in Industrial Water Samples</h2> | ||
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+ | After visiting the Armstrong ceiling tile manufacturing plant, 4 possible problematic areas were identified; aeration basin, secondary clarifier, primary clarifier, and thickener. 6 water samples were collected from these 4 locations; </p1> | ||
+ | <p1>1) aeration basin </p1> | ||
+ | <p1>2) secondary clarifier </p1> | ||
+ | <p1>3) primary clarifier to the equalization basin</p1> | ||
+ | <p1>4) thickener to primary clarifier </p1> | ||
+ | <p1>5) primary clarifier to dry broke</p1> | ||
+ | <p1>6) thickener</p1> | ||
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<IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/0/06/BroadRun_Testing_fig5.png" style="width: 750px;height: 412px;"> | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/0/06/BroadRun_Testing_fig5.png" style="width: 750px;height: 412px;"> | ||
+ | <p1><i>Industrial water samples from a ceiling tile manufacturing plant.</p1> </i> | ||
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+ | <p1>The six water samples were first tested to determine starch levels. The aeration basin, secondary clarifier, and primary clarifier to equalization basin samples did not contain a detectable level of starch. The thickener to primary clarifier and thickener to dry broke samples contained a small amount of starch, approximately 0.32% and 0.45%, respectively. The primary clarifier to dry broke contained a much higher percentage of starch, approximately 1.3%. Thus, the prototype testing was run with the following three samples; primary clarifier to dry broke, thickener to primary clarifier, and thickener to dry broke. </p1> | ||
+ | <p1>Construct 3 was found to be most effective in the previous test, thus this genetically modified yeast strain was used in prototype testing. Yeast cultures were mixed into the industrial water sample in a 1:8 ratio, with a total volume of 250ml. To account for starch degradation from other organisms in the water sample, a control without yeast cells was run. The control contained a 1:8 ratio of YPD media (without yeast cells) to industrial water sample. The mechanical agitation was simulated by adding a magnetic stirrer bar and placing the beaker onto a stirrer plate, which kept cells, starch, and other organic compounds suspended in the water sample evenly mixed throughout, as in the ceiling tile plant.</p1> | ||
<h4>Prototype</h4> | <h4>Prototype</h4> | ||
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− | + | <p1>In order to simulate the physical conditions of the plant, a prototype was created. Dissolved oxygen levels were simulated by continuously aerating the samples, mimicking the large blowers used in the ceiling tile plant. In addition to aeration, wastewater and process water in the plant is mechanically agitated, usually with large rotating rakes in the clarifiers and thickeners. </p1> | |
<IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/0/01/BroadRun_Testing_fig6.png" style="width: 750px;height: 293px;"> | <IMG class = "displayed" src="https://static.igem.org/mediawiki/2016/0/01/BroadRun_Testing_fig6.png" style="width: 750px;height: 293px;"> | ||
− | < | + | <p1><i>Setup of prototype with stirrer plates and air pump. </p1></i> |
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+ | <p1> Samples were measured at 6, 24, 48, and 72 hours. The same starch measurement protocol as in the long term starch degradation test was used. </p1> | ||
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<h4>Testing in YPD Media</h4> | <h4>Testing in YPD Media</h4> | ||
− | + | <p1>Two cell growth experiments were run; cell growth in standard yeast media (YPD media) and cell growth in starch media. </p1> | |
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+ | <h2>Cell Growth in YPD Media </h2> | ||
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+ | <p1>This experiment was run to determine parameters for creating a mathematical model. It was also run to determine if the genetic modifications to the yeast and the increased metabolic strain of constitutively producing amylase enzymes would have an effect on cell growth rates and glucose consumption. YPD media (standard liquid yeast media) was used as a glucose rich substrate. </p1> | ||
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+ | <p1>Due to logistical constraints (shortage of stirrer plates), only 2 cultures were run, wildtype yeast as a control, and Construct 2 yeast (simply referred to as ‘Genetically modified yeast’). 30mL of YPD media was added to 50mL flasks then inoculated with the 2 yeast strains. To prevent settling of the cells, cell cultures were placed on stirrer plates with stirrer bars. Cell cultures were diluted 1:4 with water before measuring in the spectrophotometer for optical density. A standard blood glucose meter was used to measure glucose in the samples, after diluting the samples 1:15. </p1> | ||
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<h4>Testing in Starch Media</h4> | <h4>Testing in Starch Media</h4> | ||
− | + | <p1>To gain insight on the ability of the genetically modified yeast to grow in substrate that contains starch, but no glucose, cell growth testing was completed in a starch media. The media contained starch, the carbon source for the yeast, and heat killed wildtype yeast, a nitrogen and amino acid source. 30mL of media was composed of: 29mL of 1% starch and 1mL of heat killed wildtype yeast. This media was added to a 50mL flask and then inoculated with Construct 2 yeast. Stir bars were added, and the flasks were left on stir plates for 192 hours (8 days). The yeast culture was measured at hours: 0, 24, 48, 54, 72, 78, 168, and 192. </p1> | |
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+ | <p1>The rationale behind this test was that the genetically modified yeast, which have the unique ability to degrade starch, would be able convert the surrounding starch into glucose, and thus be able to survive in the absence of a direct glucose source. </p1> | ||
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Latest revision as of 23:08, 19 October 2016
.MathJax nobr>span.math>span{border-left-width:0 !important};
Methods
Amplification PCR
Restriction Digest
Restriction Digest Purification
Ligation
E.coli Transformation
Colony PCR
Miniprep
Yeast Transformation
Testing
Phase 1: Testing in Known Starch Concentrations
Short Term Testing
- 1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution.
- 2. Add 0.5mL of this yeast/starch solution to a cuvette.
- 3. Add 0.3mL of 1M HCL to the cuvette.
- 4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%.
- 5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well.
- 6. Measure absorbance using the spectrophotometer.
Long Term Testing
In this test, the 4 yeast strains were combined with 2% starch in a 1:8 ratio of yeast to starch. This lower ratio and higher concentration of starch was to ensure that the yeast would not break down all of the starch within just a few hours. This solution was aerated using an air pump and tubing, to keep oxygen levels in the sample high. Setup of long term starch degradation testing. The 4th flask, with wildtype culture, is not pictured. Measurements were made every 24 hours, for 72 hours, using the following protocol: 1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. 2. Add 0.5mL of this yeast/starch solution to a cuvette. 3. Add 3.5 mL of water and 0.3mL of 1M HCL to the cuvette. 4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. 5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. 6. Measure absorbance using the spectrophotometer. Phase 2: Testing in Industrial Water Samples
After visiting the Armstrong ceiling tile manufacturing plant, 4 possible problematic areas were identified; aeration basin, secondary clarifier, primary clarifier, and thickener. 6 water samples were collected from these 4 locations;1) aeration basin 2) secondary clarifier 3) primary clarifier to the equalization basin 4) thickener to primary clarifier 5) primary clarifier to dry broke 6) thickener Industrial water samples from a ceiling tile manufacturing plant. The six water samples were first tested to determine starch levels. The aeration basin, secondary clarifier, and primary clarifier to equalization basin samples did not contain a detectable level of starch. The thickener to primary clarifier and thickener to dry broke samples contained a small amount of starch, approximately 0.32% and 0.45%, respectively. The primary clarifier to dry broke contained a much higher percentage of starch, approximately 1.3%. Thus, the prototype testing was run with the following three samples; primary clarifier to dry broke, thickener to primary clarifier, and thickener to dry broke. Construct 3 was found to be most effective in the previous test, thus this genetically modified yeast strain was used in prototype testing. Yeast cultures were mixed into the industrial water sample in a 1:8 ratio, with a total volume of 250ml. To account for starch degradation from other organisms in the water sample, a control without yeast cells was run. The control contained a 1:8 ratio of YPD media (without yeast cells) to industrial water sample. The mechanical agitation was simulated by adding a magnetic stirrer bar and placing the beaker onto a stirrer plate, which kept cells, starch, and other organic compounds suspended in the water sample evenly mixed throughout, as in the ceiling tile plant. Prototype
In order to simulate the physical conditions of the plant, a prototype was created. Dissolved oxygen levels were simulated by continuously aerating the samples, mimicking the large blowers used in the ceiling tile plant. In addition to aeration, wastewater and process water in the plant is mechanically agitated, usually with large rotating rakes in the clarifiers and thickeners. Setup of prototype with stirrer plates and air pump. Samples were measured at 6, 24, 48, and 72 hours. The same starch measurement protocol as in the long term starch degradation test was used. Phase 3: Cell Growth Testing
Testing in YPD Media
Two cell growth experiments were run; cell growth in standard yeast media (YPD media) and cell growth in starch media. Cell Growth in YPD Media
This experiment was run to determine parameters for creating a mathematical model. It was also run to determine if the genetic modifications to the yeast and the increased metabolic strain of constitutively producing amylase enzymes would have an effect on cell growth rates and glucose consumption. YPD media (standard liquid yeast media) was used as a glucose rich substrate. Due to logistical constraints (shortage of stirrer plates), only 2 cultures were run, wildtype yeast as a control, and Construct 2 yeast (simply referred to as ‘Genetically modified yeast’). 30mL of YPD media was added to 50mL flasks then inoculated with the 2 yeast strains. To prevent settling of the cells, cell cultures were placed on stirrer plates with stirrer bars. Cell cultures were diluted 1:4 with water before measuring in the spectrophotometer for optical density. A standard blood glucose meter was used to measure glucose in the samples, after diluting the samples 1:15. Testing in Starch Media
To gain insight on the ability of the genetically modified yeast to grow in substrate that contains starch, but no glucose, cell growth testing was completed in a starch media. The media contained starch, the carbon source for the yeast, and heat killed wildtype yeast, a nitrogen and amino acid source. 30mL of media was composed of: 29mL of 1% starch and 1mL of heat killed wildtype yeast. This media was added to a 50mL flask and then inoculated with Construct 2 yeast. Stir bars were added, and the flasks were left on stir plates for 192 hours (8 days). The yeast culture was measured at hours: 0, 24, 48, 54, 72, 78, 168, and 192. The rationale behind this test was that the genetically modified yeast, which have the unique ability to degrade starch, would be able convert the surrounding starch into glucose, and thus be able to survive in the absence of a direct glucose source.