Team:Concordia/Notebook/NanoLyt
Nanoshell Attachment Method and Lyticase Test Notebook
AUGUST 8
Preparation of Lyticase Solution
Measured 2.1mg of lyticase powder (≥2000 units/mg)
Added the lyticase powder to 500µL of 100% glycerol and 500µL of 1X TE buffer to create a 1mL stock solution of lyticase.
Stored in -20°C freezer
AUGUST 9
Grew an overnight culture of leucine deficient Saccharomyces cerevisiae.
1. Measured 0D600 of cells = 5.04
2. Transferred 2mL of cells to a 2mL Eppendorf tube and centrifuge at 5000rpm for 20 minutes to collect cells.
3. Decant supernatant, resuspend in 1mL of PBS
4. Centrifuge at 5000rpm for 20 minutes
5. Decant supernatant, resuspend in 1mL of PBS
6. Set up 4 tubes
A: 200µL of yeast cells + 1.8mL of gold nanoparticles coated w/ L-cysteine.
B: 200µL of L-cysteine coated yeast cells + 1.8mL of gold nanoparticles coated w/ L-cysteine
C: 150µL of L-cysteine coated yeast cells + 1.9mL of gold nanoparticles coated w/ L-cysteine
D: 50µL of L-cysteine coated yeast cells + 1.95mL of gold nanoparticles coated w/ L-cysteine
7. Incubate at room temperature for half an hour
8. Centrifuge at 4000rpm for 15 minutes
9. Decant supernatant and resuspend in 1mL of PBS
Took these images to a microscope and observed under dark-field and bright-field. The camera attachment on the microscope did not allow dark-field pictures, so the images are only of bright-field.
Control:
Samples from Tube B:
Samples from Tube C:
AUGUST 17
Lyticase Test: Testing Different Conditions for Cells Death
Purpose: To test whether yeast cells inoculated in TE buffer or 0.85% NaCl will lyse once lyticase has been added.
Prepared 0.85% NaCl:
Dissolved 8.5g of NaCl pellets in 10mL of water
Inoculation of cells:
Grew an overnight culture of Saccharomyces cerevisiae on YPD media.
OD of overnight culture at 600nm: 11.34
We want to show the drop in OD over a long period of time, so we want to start at an OD of 1.
We want a final volume of 4mL.
C1V1 = C2V2
(11.34)(V1) = (0.1)(4mL)
V1 = 353µL
Added 353µL of original cell culture (OD=11.34) to 3.647mL of 1X TE buffer in one tube. Repeat in 3 other tubes.
Added 353µL of original cell culture (OD=11.34) to 3.647mL of 0.85% NaCl in one tube. Repeat in 3 other tubes.
Initial OD600 of all tubes = 1
There are 4 tubes containing TE buffer and cells and 4 tubes containing NaCl and cells. In 2 of the tubes containing TE buffer, added 10 µL of the lyticase stock solution prepared on August 9th.
In 2 of the 4 tubes containing NaCl and cells, added 10 µL of the lyticase stock solution.
Incubated the samples at 30°C. Took samples from each tube after 1 hour and measured the OD600 and cell concentration using a hemocytometer.
From these results we can determine that when lyticase is added to cells in TE buffer, it leads to cell lyses.
The NaCl shows an increase in OD instead of a decrease but this is likely due to the fact that we did not include a washing step before inoculating the cells in NaCl. So a little bit of YPD media was transferred along with the cells and they had nutrients to grow. However, the cell count data shows a decrease in the OD. In future experiments, we will add lyticase to cell solutions in TE buffer to see if cells are being lysed or not.
AUGUST 25
Lyticase Test on Gold Nanoshell Attachment using Bought Nanoparticles
Grew an overnight culture of leucine deficient Saccharomyces cerevisiae.
1. Measured 0D600 of cells = 10.4
2. Transferred 2mL of cells to a 2mL Eppendorf tube and centrifuge at 5000rpm for 20 minutes to collect cells.
3. Decant supernatant, resuspend in 1mL of PBS
4. Centrifuge at 5000rpm for 20 minutes
5. Decant supernatant, resuspend in 1mL of PBS
6. Set up 10 tubes according to the following table:
*Vol yeast corresponds to the volume of cell solution taken from the tube containing PBS/yeast (Step 5)
7. Incubate at room temperature for half an hour
8. Centrifuge at 4000rpm for 15 minutes
9. Resuspend in 1mL of 1X TE buffer
10. To one set of tubes, add 10µL of lyticase
11. Using a Tecan SunriseTM machine, measure OD600 of all samples every 20 minutes for a total of 5 hours. The machine incubates the samples at 37°C.
Results:
There were 16 time points at which the OD600 was measured by the machine. Every 20 minutes for 5 hours.
This graph compared yeast in TE buffer with or without lyticase. When lyticase is present, the yeast cells decrease in OD. Without lyticase, the OD is mostly constant with a slight increase.
One of the issues with this data is that we did not start at a high enough OD, so we do not see enough of a drop in OD.
This graph compares yeast without nanoparticles to yeast coated with nanoparticles. Lyticase was added to both. There doesn’t seem to be a difference in the drop in OD for these 2. The solution containing the nanoparticles starts at a higher OD even though the number of cells in both samples is equal. This is because the presence of the nanoparticles increases the absorbance that is read by the machine. To accurately interpret results, we should look at how much the OD has dropped in each case.
This graph shows yeast cells coated in nanoparticles. Lyticase was added to one of the samples but not the other. This graph looks similar to the Effects of Nanoparticles on Yeast graph, which implies that the nanoshell is not protecting the cells from lyticase.
*For future experiments, start at a higher OD. Try attaching with nanoparticles we synthesized.
AUGUST 30
Lyticase Test on Nanoshell Attachment using Gold Nanoparticles Synthesized through Martin Method
Followed nanoshell attachment and lyticase test protocol from August 25th (refer above). The only difference was that we started with a higher OD and used nanoparticles synthesized through Martin method.
The values in the first column of the table above correspond to time in hours. The OD600 was measured every 20 minutes for 5 hours, which is why some numbers in this column appear as fractions of an hour.
The graph above only includes data from samples that did not contain lyticase. It compares nanoparticle coated yeast cells with control cells lacking nanoparticles. The nanoparticle coated cells show a similar pattern of OD as yeast cells without nanoparticles. This implies that nanoparticles may not be harming the cells.
This graph compares nanoparticle coated yeast with control yeast lacking nanoparticles. The addition of lyticase causes the OD to drop in either case. The nanoshell doesn’t appear to be protecting the cells according to this data. However, this data is somewhat inconclusive because the OD of the samples containing lyticase drops as soon as the lyticase is added, even before the first OD reading determined from the machine. This is why the starting OD of yeast with lyticase and without lyticase is different.
The graph above displays only the initial OD and the final OD measured. The addition of lyticase leads to a drastic decrease in OD in both cases. However, the initial OD of Yeast w/out lyticase and Yeast w/ lyticase should be the same. As stated earlier, this is because the lyticase drops the OD of the yeast before the initial OD is measured by the machine. In future experiments, this can be avoided by measuring the initial OD of the samples before the lyticase is added.
SEPTEMBER 8
Lyticase Test Varying the Concentration of Lyticase
Followed nanoshell attachment and lyticase protocol from August 25th (refer above). Used nanoparticles synthesized from the Martin method. Instead of preparing samples using varying percentages of nanoparticles, only looked at samples either containing no nanoparticles or 80% nanoparticles.
Used different dilutions of lyticase.
Stock solution of lyticase (from August 9th) contained 2.1 mg of ≥2000 units/mg lyticase powder in 500µL 100% glycerol and 500µL 1X TE buffer. This created a 1mL stock solution.
For 10-fold dilution:
Transferred 50µL from stock lyticase solution to new Eppendorf tube. Added 225µL of 100% glycerol and 225 µL of 1X TE buffer. This creates a 500 µL solution that is diluted 10-fold from the stock.
For 50-fold dilution:
Transferred 10 µL from stock lyticase solution to new Eppendorf tube. Added 245µL of 100% glycerol and 245 µL of 1X TE buffer. This creates a 500 µL solution that is diluted 50-fold from the stock.
Measuring OD:
Had 4 samples of yeast without nanoparticles. The first sample did not have lyticase added to it. The other 3 samples had either undiluted, 10-fold diluted or 50-fold diluted lyticase added. Then the OD at 600nm was measured every 20 minutes for 5 hours.
Had 4 samples of nanoparticle coated yeast. The first sample did not have lyticase added to it. The other 3 samples had either undiluted, 10-fold diluted or 50-fold diluted lyticase added. Then the OD at 600nm was measured every 20 minutes for 5 hours.
Instead of incubating at 37°C as in previous lyticase tests, incubated the samples at 30°C this time.
Results
The first column represents time points taken every 20 minutes for 5 hours (for a total of 16 time points)
The nanoparticle coated yeast samples that contained 80% nanoparticles seemed to drop in OD and then increase again. This pattern is something that was not observed when the samples were incubated at 37°C. The most significant difference between nanoparticle coated yeast and yeast without nanoparticles appears to be when 50-fold diluted lyticase is added. This can be seen more clearly in the graph below which only contains the initial and final OD of the samples.
From this attempt, we’ve concluded that 50-fold diluted lyticase should be used in future lyticase tests because it seems to yield the best results.
OCTOBER 6
Nanoshell Attachment of Gold Nanoparticles Using Martin Method and Garlic Nanoparticles
Same procedure as previously written.
The samples were brought to a dark field microscope with a camera attachment.
Control Yeast Cells:
Martin Nanoparticle Coated Yeast:
Martin Nanoparticle Coated Yeast:
Garlic Nanoparticle Coated Yeast:
