Genetic constructs synthesized by IDT were reconstituted in 100µl of TE buffer (recipe below) and incubated at 50˚C for 20 minutes. Primers for amplification PCR were resuspended in sterile water.
TE Buffer recipe
Makes 1mL of TE BufferCombine the following in a microcentrifuge tube:
2µl EDTA (.5M, pH 8.0)10µl Tris (1M, pH 8.0) 988µl water
Amplification PCR
Protocol
1. Make Master Mix (recipe below)
2. Aliquot 50µl into each tube. 3. Add primers, DNA, and water as per protocol below.
1. Combine reagents as per below, adding water first and enzymes last. Keep enzymes chilled.
2. Incubate at 37˚C for 60 minutes.
3. Heat kill enzymes at 60˚C for 15 minutes.
4. Purify digest products using a PCR purfication spin kit.
Digest of Amplification PCR Products
Reaction 1
Reaction 2
Reaction 3
Reaction 4
Reaction 5
Reaction 6
DNA (µl)
20 of GC1
20 of GC2
20 of GC3
20 of GC1
20 of GC2
20 of GC3
Enzyme 1 (µl)
1 of KpnI
1 of KpnI
1 of EcoRI
1 of EcoRI
1 of EcoRI
1 of EcoRI
Enzyme 2 (µl)
1 of SpeI
1 of SpeI
1 of SpeI
1 of PstI
1 of PstI
1 of PstI
Cut Smart Buffer (µl)
10
10
10
10
10
10
Water (µl)
63
63
63
63
63
63
Digest of Vectors
pRS426
pAG36
pSB1c3
DNA (µl)
5 of pRS426
5 of pAG36
5 of pSB1c3
Enzyme 1 (µl)
1 of EcoRI
1 of KpnI
1 of EcoRI
Enzyme 2 (µl)
1 of SpeI
1 of SpeI
1 of PstI
Cut Smart Buffer (µl)
5
5
5
Water (µl)
38
38
38
Week 3
Ligation
After digesting and purifying our vectors and constructs, we ligated the two DNA samples together. For each reaction, reagents were mixed as follows.
Ligation Reaction
Reagent
Volume (µl)
Vector
2
Insert
6
Ligase Buffer
1
Ligase
2
*Note: Our DNA construct samples were dilute, which was a higher volume was used, and no water was used.
E.coli Heat Shock Transformation
After ligation, the ligated DNA samples were transformed to E.coli and plated on LB plates with antibiotics (ampicillin for 2 yeast vectors, chloramphenicol for pSB1c3 vector), as per the protocol below.
1. Thaw competent cells on ice.
2. Add 50µl of cells to sterile microcentrifuge tubes.
3. Add 2-5µl of ligation reaction to each tube and finger vortex.
4. Incubate on ice for 20 minutes. Begin heating up the water bath to 42˚C at this time.
5. Add 1mL of SOC recovery media to glass culture tubes and label.
6. Heat shock cells by placing in 42˚C water bath for 45 seconds.
7. Immediately put the tubes on ice for 5 minutes.
8. Add the cells and DNA mixture to each glass culture tube with SOC media. Incubate for 1-2 hours at 37˚C.
9. Transfer cultures to microcentrifuge tubes and centrifuge for 3 minutes at 5000rpm.
10. Without disturbing the pellet, remove 700µl of supernatant. Resuspend the pellet in the remaining supernatant.
11. Plate 100-200µl onto LB plates with the appropriate antibiotics. Spread with sterile glass beads or sterile spreader.
12. Incubate 12-24 hours overnight at 37˚C
Week 4
Colony PCR
After bacterial transformation, we ran colony PCR to determine which of the colonies contained the plasmid with the desired insert, as per the protocol below.
1. Make Master Mix (recipe below)
2. Aliquot 20µl into each tube
3. Use a sterile toothpick to choose a colony from the bacterial plate. Dip the toothpick into one of the PCR tubes and swirl it. Using the same toothpick, either streak the toothpick onto a labeled LB plate or mix into a culture tube with fresh LB broth.
4. Repeat step 3 for all the remaining colonies.
5. For a negative control, add 1µl of sterile water into a tube with 20µl of the master mix.
6. Place the tubes into the PCR machine and set it to run with the protocol below.
Master Mix Recipe (makes 400µl, enough for 20 of 20µl reactions)
Reagent
Concentration
Volume
Forward Primer
100µM
4µl
Reverse Primer
100µM
4µl
dNTPs
10mM
7.5µl
Taq Buffer
10X
40µl
Taq
N/A
2µl
Water
N/A
288µl
After colony PCR confirmed which colonies contained our DNA insert, we miniprepped those liquid cultures using a spin column miniprep kit. For the colonies with the psb1C3 plasmid, we mailed those miniprepped samples to iGEM, to add to their registry. For the colonies with the yeast plasmids, we transformed those samples to S.cerevisiae.
Week 5
Yeast Transformation
In order to transform our miniprepped plasmids into S.cerevisiae, we first had to prep the cells for transformation, a process that took several days. Our yeast plasmids used the selectable marker gene URA3, so we used the corresponding yeast strain. This means the URA3 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. Preparation of Yeast Cultures for Transformation
1. The yeast strain was inoculated into 25mL of liquid 2X YPAD media and incubated overnight at 30˚C.
2. The titer of cell culture was determined using a hemacytometer. 2.5 x 10^9 cells were added to 500mL of 2X YPAD media.
3. Incubate flask at 30˚C until the cell titer is 2 x 10^7 or higher, about 4 hours.
4. Centrifuge the cells at 3000rpm for 5 minutes. Remove the YPAD broth without disturbing the pellet.
5. Wash the cells in half the volume of sterile water and resuspend in .01 volume of sterile water. (we divided our 500mL culture into 14mL tubes; thus, wash in 7mL of water, and re-suspend in 140µl of water)
6. Transfer to microcentrifuge tubes and centrifuge again for 5 minutes.
7. Resuspend the cell pellet in 140µl of frozen competent cell solution (5%w/v glycerol, 10%v/v DMSO).
8. Dispense 50µl samples into microcentrifuge tubes. Place the tubes into a styrofoam container.
9. Freeze the cells overnight in a -80˚C freezer overnight.
Yeast Transformation
1. Thaw cells in a 37˚C water bath for 30s.
2. Centrifuge at 13000rpm for 2 minutes and remove supernatant.
3. Boil carrier DNA at 100˚C for 5 minutes to denature it.
4. For each tube of competent yeast cells, add the reagents in the following order:
- Polyethylene glycol (50%w/v) - 260µl
- Lithium acetate (1.0 M) - 36µl
- Carrier DNA - 50µl
- Plasmid DNA - 14µl
4. Include one extra tube with 14µl of sterile water in place of the plasmid DNA for a negative control.
5. Incubate in a 42˚C water bath for 30 minutes.
6. Pellet the cells by centrifuging at 13000rpm for 1 minute and remove supernatant.
7. Resuspend well in 1mL of sterile water.
8. Plate 100µl and 200µl of cell suspension onto uracil deficient media plates. Spread with sterile glass beads.
9. Incubate plates at 30˚C for 3 days.
After incubation, colonies were inoculated into liquid YPD media and these liquid cultures were used in the testing phase of the project.
Week 6
We decided to first test the yeast in a solution with a known starch concentration and test the breakdown of starch over a period of 6 hours. We tested 4 different yeast strains: three genetically modified yeast (three different constructs) and wildtype yeast as a control. Before actually adding starch to the yeast cultures, we needed to ensure that all the cultures were at the same cell density. We did so using the spectrophotometer. We created a protocol as follows: Cell Density Measurement
1. Briefly mix cultures to ensure all cells are evenly mixed throughout the media.
2. Blank the spectrophotometer by setting the wavelength to 600nm, inserting a blank cuvette with YPAD media (no cells), and setting transmission to 100%.
3. Add 4mL of each of the 4 liquid cultures to cuvettes and measure absorbance.
4. Dilute cultures accordingly, to achieve an absorbance level of ~1.6
Once all of the yeast cultures were at the same cell density, we could move onto the starch degradation measurements. Starch Degradation Measurements
1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. The starch used was standard lab grade potato starch.
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.
We used this measurement as ‘Time Zero’, that is when no starch has been broken down yet. The 4 tubes of yeast/starch were kept in an incubator. Every hour, 0.5mL was taken out and tested in the spectrophotometer, using the same method as above. The spectrophotometer was re-blanked each hour when a measurement was made, to ensure accurate readings. This was done for 6 hours. We stopped at 6 hours because at that point, the spectrophotometer was giving out readings of ~0.1 absorbance level. Below 0.1, the spectrophotometer’s readings are not as accurate.
Week 7
Long Term Starch Degradation Test
Having confirmed that the yeast were producing amylase and were able to degrade starch in the previous test, we moved on to testing starch degradation over several days. For this set of experiments, we used a higher concentration of starch (2%), and a lower ratio of yeast to starch (1:8). We also used an air pump to aerate the samples. Set up 1. Check cell density of cultures to ensure all are at the same density. 2. Combine 25mL of yeast culture with 225 mL of 2% starch solution in a 250mL flask. Repeat for all 4 yeast types (3 genetically engineered, 1 wildtype as control)3. Aerate samples using an air pump and tubing with a sterile 1mL pipette in the flask.
Starch Degradation Measurement
1. Add 0.5mL of the yeast/starch solution to a cuvette.
2. Add 3.5 mL of water, and 0.3mL of 1M HCL to the cuvette.
3. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%.
4. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well.
5. Measure absorbance using the spectrophotometer.
Samples were taken at 0, 24, 48, and 72 hours.
Week 8
Industrial Water Sample Testing
6 Samples were obtained from Armstrong’s plant, to test the ability of the yeast to degrade starch in an industrial water sample.
1. Aeration Basin
2. Secondary Clarifier
3. Primary Clarifier to Equalization Basin
4. Primary Clarifier to Dry Broke
5. Primary Clarifier to Thickener
6. Thickener
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 primary clarifier to thickener and thickener samples contained a small amount of starch. The primary clarifier to dry broke contained a much higher percentage of starch. Thus, we ran our prototype testing with these three samples; primary clarifier to dry broke, primary clarifier to thickener, and thickener. To account for starch degradation from organisms already in the water, we ran a control without yeast cells. 25mL of sterile YPD broth was substituted for yeast culture in the control.In order to simulate the physical conditions of the plant, we created a prototype. We simulated the DO levels by continuously aerating the samples. The mechanical agitation was simulated by adding a stirrer bar, which kept substrates in the sample evenly mixed throughout, as in the ceiling tile plant.
Setup
1. Check cell density of cultures to ensure all are at the same density. 2. Combine 25mL of the yeast strain with construct 3 with 225 mL of water sample from in a 250mL flask. 3. Combine 25mL of YPD broth (for the control) in 225mL of water sample in a 250mL flask. 4. Place on stirrer plate with stirrer bar to provide mechanical agitation. 5. Aerate samples using an air pump and tubing with a 1mL pipette in the flask.
The same testing protocol as before was used to measure starch levels. Samples were taken at 0, 6, 24, 48, and 72 hours.
Week 9
Cell Density Testing
In order to have more experimental data for creating a mathematical model, cell growth tests were run. A test was also run to see if the genetically engineered yeast could survive on starch and non-living yeast, in order to simulate conditions of the plant. Setup
Cell Growth in standard YPD media 1. Add 30mL of sterile YPD media each to two 50mL flasks2. Inoculate with yeast, one with wildtype, the other with genetically modified yeast. 3. Add sterile stir bars and place on stirrer. Cell Growth in Starch environment1. Make 1% starch solution2. Add 1 packet of wildtype yeast to 50mL of sterile water and bring to a boil on a hot plate. Let boil for 5 minutes to heat kill all the yeast. 3. After cooling, add 1mL of boiled yeast and 29mL of starch solution to a 50mL flask. 4. Inoculate with genetically modified yeast. 5. Add sterile stir bars and place on stirrer. Cell Density Measurements
1. Blank the spectrophotometer by setting the wavelength to 600nm, using a blank cuvette of 1mL YPAD media (no cells) and 3mL water. For the culture in starch media, a solution of the starch and boiled yeast without any cells was used to blank the spectrophotometer. 2. Add 1mL of each cell culture and 3mL of water to cuvettes. Measure absorbance. 3. To measure glucose, 14µl of water was pipetted onto a piece of parafilm and 1µl of the cell and YPD mixture was added. 4. The diluted cell culture/YPD mixture was measured using a standard blood glucose meter.
Week 10
Calibration Curves
A total of calibration curves were created, to determine concentrations in the unknown samples in the experiments; glucose, and cell density. Glucose Calibration curve For the glucose calibration curve, dilutions of the YPD yeast media were made and measured with a standard blood glucose meter. The following dilutions were made: 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, and 1:70, where 1:5 means 1µl of YPD and 4µl of water.These dilutions represent the range of the glucose meter, a 1:4 dilution was too high a glucose concentration for the meter, and a 1:80 dilution was too low a glucose concentration for the meter. Cell density measurements For the cell density measurements, 5 dilutions of a yeast culture were made; 1:2, 1:4, 1:8, 1:16, and 1:32. Each dilution was measured in the spectrophotometer at 600nm.
