Line 77: | Line 77: | ||
<h4>Miniprep </h4> | <h4>Miniprep </h4> | ||
− | + | ||
<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> | <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> | ||
− | Yeast Transformation | + | <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> | <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> | ||
Line 109: | Line 109: | ||
<p1>Starch measurements were made using the following protocol: </p1> | <p1>Starch measurements were made using the following protocol: </p1> | ||
<ul style="list-style-type:none"> | <ul style="list-style-type:none"> | ||
− | <li>1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. | + | <li style="font-size:20px";>1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. |
− | <li>2. Add 0.5mL of this yeast/starch solution to a cuvette. | + | <li style="font-size:20px";>2. Add 0.5mL of this yeast/starch solution to a cuvette. |
− | <li>3. Add 0.3mL of 1M HCL to the cuvette. | + | <li style="font-size:20px";>3. Add 0.3mL of 1M HCL to the cuvette. |
− | <li>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. | + | <li style="font-size:20px";>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. |
− | <li>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. | + | <li style="font-size:20px";>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. |
− | <li>6. Measure absorbance using the spectrophotometer. | + | <li style="font-size:20px";>6. Measure absorbance using the spectrophotometer. |
Line 130: | Line 130: | ||
<p1>Measurements were made every 24 hours, for 72 hours, using the following protocol: </p1> | <p1>Measurements were made every 24 hours, for 72 hours, using the following protocol: </p1> | ||
<ul style="list-style-type:none"> | <ul style="list-style-type:none"> | ||
− | <li2>1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. | + | <li2 style="font-size:20px"; >1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. |
− | <li2>2. Add 0.5mL of this yeast/starch solution to a cuvette. | + | <li2 style="font-size:20px";>2. Add 0.5mL of this yeast/starch solution to a cuvette. |
− | <li2>3. Add 3.5 mL of water and 0.3mL of 1M HCL to the cuvette. | + | <li2 style="font-size:20px";>3. Add 3.5 mL of water and 0.3mL of 1M HCL to the cuvette. |
− | <li2>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. | + | <li2 style="font-size:20px";>4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%. |
− | <li2>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. | + | <li2 style="font-size:20px";>5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well. |
− | <li2>6. Measure absorbance using the spectrophotometer. | + | <li2 style="font-size:20px";>6. Measure absorbance using the spectrophotometer. |
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.