NOVEMBER 16th 2023
pSB1A3-ScHIS3 Transformation, Colony PCR and Incubation
Since we needed to construct the plasmid for pRS423, we transformed pSB1A3-ScHIS3 into our Escherichia coli strain, which was performed through heat shock. We resuspended the contents of well N1 and transformed it into our competent cells, testing any visible colonies by colony PCR, so that we could plate these transformed cells on Luria-Bertani (LB)-Amp plates, incubated overnight.
NOVEMBER 17th 2023
pRS423 Miniprep, Gibson Assembly and pRS426 Purification and Cultivation
We prepared minipreps, through purification of the plasmids from our cultures with Cytiva’s PlasmidPrep kit, in order to use these minipreps as template DNA for ScHIS3 marker amplification with the Gibson Assembly method. We needed to do the same for pRS426, which was already available in the lab. Then pRS426 was purified and E. coli strain carrying the empty closed plasmid was cultivated in LB supplemented with ampicillin overnight.
NOVEMBER 18th 2023
pRS426 Miniprep, ScHIS3 PCR and Gel electrophoresis
We purified the plasmids cultivated the day before using Cytiva’s PlasmidPrep kit and quantified this miniprep. In order to be able to begin the construction of our pRS423, we needed to amplify both ScHIS3 and pRS426. With the goal of amplifying the ScHIS3 selection mark, we performed a PCR with the primers designed for ScHIS3 marker sequence. We also ran an agarose gel electrophoresis to check our amplification and, apparently, it worked out.
FEBRUARY 27th 2024
pRS426 Digestion
Before amplifying pRS426, we concluded it would be best to digest it first, since the digestion prior to PCR makes it easier for the denaturing and annealing of primers, and would yield a better amplification. We digested our pRS426 miniprep with Ndel.
FEBRUARY 28th 2024
pRS426 PCR and Gel Electrophoresis
We used the digestion from the day before as template for our plasmid PCR with primers designed based on the pRS426 sequence around the URA3 marker. After the procedure, we ran agarose gel electrophoresis to confirm the amplification and confirmed it.
MARCH 1st 2024
ScHIS3 Purification, Gibson Assembly and Incubation
Since our amplifications were the appropriate size for cloning, we purified our fragments using Cytiva’s GFX PCR purification kit, quantified the purified fragments and proceeded to cloning through Gibson Assembly, since it was the fastest and most reliable method we had. We used our purified ScHIS3 fragments, purified pRS backbone and our homebrew Gibson Assembly Master Mix. This reaction was set up on ice, mixed in a PCR tube and incubated.
MARCH 2nd 2024
E. coli Transformation and Incubation
After the reaction incubation, we transformed this reaction mix into competent E. coli cells, so that we could incubate them overnight in LB-Amp plates.
MARCH 3rd 2024
E. coli Colony PCR, Gel Electrophoresis and Incubation
The E. coli colonies were tested by colony PCR. We ran an agarose gel electrophoresis. The results found many positive colonies. These positive colonies were inoculated into fresh LB-Amp and incubated overnight, for plasmid amplification.
APRIL 3rd 2024
IDT DNA Treatment, PCR and Gel Electrophoresis
We got our cassettes as gBlocks from IDT. Aiming to amplify each fragment we got from IDT, we resuspended and treated the dried DNA in TE buffer and attempted to perform PCRs using these resuspended fragments directly as templates with primers designed for each fragment adding homologies for Gibson Assembly. To check our amplification, we ran agarose gel electrophoresis, but we weren’t able to detect bands on the gel.
APRIL 5th 2024
IDT Mini Gibson Assembly
Since gBlocks are short fragments with overlaps, we concluded our best alternative to try amplification again would be Mini Gibson Assembly, followed by PCRs. We took the synthesis and incubated all reactions with our homemade GA Master Mix. These GA reactions were used directly as templates in PCR and we had amplifications. Then, we purified, quantified and stored these purifications, so that they would be ready for cloning when needed.
APRIL 10th 2024
pRS423 and pRS426 Minipreps Digestions, Incubation and PCRs
Moving to plasmid amplification, we digested each miniprep of pRS423 and pRS426 with Notl prior to PCR amplification, in order to get a better yield. After incubating these reactions, the restriction enzyme was inactivated and the reactions were used as template for PCR reactions. Through agarose gel electrophoresis, we discovered only pRS426 had been amplified.
APRIL 15th 2024
pRS423 PCR and Gel Electrophoresis
We redid the pRS423 PCR, since one of the possible mistakes could have been a pipetting error. We still had no bands on agarose gel at the end of this repeated procedure.
APRIL 22nd 2024
pRS423 Minipreps and Digestions, Gel Electrophoresis and Cultivation
After checking our pRS423 minipreps and digestions on agarose gel, we confirmed that our pRS423 was not built correctly, or at the very least, degraded. To confirm if the plasmids were built incorrectly or degraded, we cultured our pRS423s glycerol stocks in LB-Amp overnight.
APRIL 25th 2024
pRS423 Minipreps and Digestions, Gel Electrophoresis and Beginning of pRS423 Reconstruction
We prepared minipreps from the cultivated pRS423 cultures and immediately ran the agarose gel to check the plasmids’ integrity. The band pattern confirmed that these plasmids were not built correctly. Because of that, we began to redo the pRS423 construction. We amplified, through PCR, and purified pRS426 and ScHIS3.
APRIL 26th 2024
pRS423 Gibson Assembly and E. coli Transformation
We proceeded with the GA reactions and transformed and plated E. coli with the Gibson Assembly products.
MAY 2nd 2024
pRS426 and ScHIS3 Colony PCR and Incubation
We performed a colony PCR, identified putative positive colonies and inoculated them into fresh LB-Amp. The cultures were incubated overnight.
MAY 3rd 2024
pRS423 Digestion and Gel Electrophoresis
We prepared the minipreps for the cultures cultivated the day before and digested them with Kpnl. We ran agarose gel and confirmed that the digested pRS423 was built correctly.
MAY 7th 2024
pRS423 Digestion, PCR and Gel Electrophoresis
Since we had the confirmation of a correct build for our plasmids, we proceeded to digest it with Notl and performed a PCR procedure to amplify it, with the digested plasmids as templates, making it ready for cloning. However, after we ran the agarose gel, we noticed that the bands we wanted weren’t detected and our plasmids were damaged.
MAY 9th 2024
pRS423 Miniprep and Incubation
We decided not to digest, since the vector was considerably damaged. We prepared a pRS423 miniprep and cultivated it in LB-Amp, incubated overnight.
MAY 10th 2024
pRS423 PCR, Gel Electrophoresis and Purification
We used the pRS423 plasmids cultivated the day before as templates for PCR reactions and ran agarose gel electrophoresis to confirm amplification. After confirmation, we purified it to prepare to clone our cassettes into our plasmids.
MAY 15th 2024
pRS426 and pRS423 Gibson Assembly and E. coli Transformation
In order to clone our pRS426 and pRS423 plasmids, we performed the Gibson Assembly reaction. Then, we transformed E. coli as we described before, to begin the process of CBDsub and CBDsyn cassettes.
MAY 18th 2024
E. coli Colony PCR
We performed a colony PCR using two primers internal to the cassette CBDsub and ran agarose gel electrophoresis to check the amplification. We observed that it didn't cover the entire construct.
MAY 24th 2024
E. coli Colony PCR and Gel Electrophoresis
For CBDsyn, we used a primer pair that covered the entire construct in the E. coli colony PCR procedure. We ran agarose gel electrophoresis, but didn’t detect bands.
MAY 28th 2024
E. coli Colony PCRs and Gel Electrophoresis
On May 15th, we incubated more E. coli than what we theoretically would use, so that we didn’t have to repeat the entire process from the beginning in case something went wrong. With our stocked colonies, both PCR procedures (with CBDsub and CBDsyn primers, respectively) were performed. This time, when we ran agarose gel electrophoresis, bands were detected successfully.
JUNE 3rd 2024
CBDsub and CBDsyn Purification and Quantification
The amplified CBDsub and CBDsyn were purified and quantified, to prepare them for cloning through Gibson Assembly.
JUNE 5th 2024
CBDsub and CBDsyn Gibson Assembly and Gel Electrophoresis
We performed the Gibson Assembly method, but neither the cassettes were cloned, since we didn’t detect bands on the agarose gel.
JUNE 11th 2024
CBDsub and CBDsyn Gibson Assembly, Gel Electrophoresis, Cultivation and Miniprep
We repeated the Gibson Assembly method for both CBDsub and CBDsyn using amplified stocked colonies from the PCR from May 28th. This time, we confirmed the cloning once we ran agarose gel electrophoresis. Any colonies that showed bands were cultured overnight in LB-Amp broth. The remaining cultures were minippreped.
JUNE 12th 2024
CBDsub and CBDsyn Digestion and Gel Electrophoresis
The purified plasmids from June 3rd were digested with Kpnl and the digestion products were applied on an agarose gel for electrophoresis. The bands on the gel showed us the cloning was successful.
JUNE 20th 2024
CBDsub and CBDsyn Digestion.
Since transformation with linear DNA is much more efficient than with plasmid DNA, we concluded that we should linearize the plasmids before beginning the transformation phase. We attempted to do so through an overnight digestion with CBDsub and CBDsyn.
JUNE 21st 2024
Gel Electrophoresis and Control Experiment
We planned to purify the digested DNA on this day, but the agarose gel electrophoresis had bands all over the place and we couldn’t use it for purification. To test if this mistake was caused due to star activity of the restriction enzymes we used, we performed a control experiment with an in-house plasmid, different from our pRS ones and left it overnight.
JUNE 22nd 2024
CBDsub and CBDsyn PCRs, Gel Electrophoresis and Purification
The control experiments faced no issues, meaning the restriction enzymes used weren’t the problem involved in the failed digestion. Then, we decided to try a different approach: design PCR primers that would cover the entire plasmid, removing the ampicillin and E. coli replication origins, as a safety measure. We performed PCR with these primers, with both plasmids, using our best minipreps for each cassette as templates. We also ran agarose gel electrophoresis and confirmed the amplification of both cassettes, then purified the linear DNAs.
AUGUST 7th 2024
Saccharomyces cerevisiae Incubation
We took our stored Saccharomyces cerevisiae and streaked it onto solid YPD-9721. Then, it was incubated for the next few days.
AUGUST 9th 2024
Yeast Growth Stimulation
We noticed our yeast was considerably small compared to other strains of Saccharomyces cerevisiae. To try to solve this problem, we added more amino acids, hoping the yeast would grow more cells, and inoculated a small amount of the yeast in liquid medium, so that the growth was stimulated even further.
AUGUST 12th 2024
Yeast Subculturing
We performed yeast subculturing, in order to reactivate our strain, since it was stocked in ultra low temperatures.
AUGUST 15th 2024
Second Round of Yeast Subculturing
The subculturing was performed again in our yeast, for the same purpose.
AUGUST 18th 2024
Third Round of Yeast Subculturing
The same procedure was performed to reactivate the Saccharomyces cerevisiae strains.
AUGUST 21st 2024
Fourth Round of Yeast Subculturing and Incubation
To keep the reactivation, the yeast was subcultured a fourth and last time, making it ready for the preparation of our competent cells. We replated the yeast on solid medium to ensure a healthy culture. Then, an isolated colony was transferred from the SC9721 strain to a flask with YPD medium and incubated.
SEPTEMBER 5th 2024
Incubation and Resuspension
After that incubation, we inoculated the culture into fresh YPD medium, repeating the incubation. The resulting culture was resuspended and we added TE/LiAc solution to those cells.
- DETERMINING THE STANDARD GROWTH CURVE FOR S. CEREVISIAE
AUGUST 7th 2024
The stock solutions of S. cerevisiae SC9721 were removed from the -80 ultrafreezer to be plated on solid YPD 9721 medium. For this process, we used 50 ul of the thawed sample and inoculated it into two petri dishes (50 ul for each dish). The plates were left in the oven at 37° C for 48 hours.
AUGUST 9th 2024
An arbitrary sample was taken from the petri dishes with the grown yeast to be inoculated into 100 ml of liquid YPD 9721 culture medium in a 500 ml erlenmeyer flask. This sample was then incubated at 30°C at 180 rpm.
Using the same petri dishes, we removed another arbitrary aliquot and resuspended it in liquid YPD medium with 70% glycerol to make a stock solution.
AUGUST 10th 2024
Due to a sudden power outage at the college, unfortunately the samples were out of the right temperature for a significant amount of time, so we didn't get good growth. Due to this problem, it was necessary to redo the cultivation steps in liquid medium according to the procedures described above
AUGUST 18th 2024
We grew the yeast again in YPD liquid medium and incubated them at 30°C at 180 rpm for 12 hours.
AUGUST 19th 2024
We measured the optical density of the culture and obtained A600 = 0.38. We chose to keep the cells growing in order to obtain better values
AUGUST 20th 2024
The optical density of the cells was measured again, this time we obtained a value of A600: 0.46 and continued with the experiments.
For the next steps, two salt solutions (NaCl) of 0.6% and 0.9% were made for resuspending the yeast cells. These solutions were autoclaved for 25 min at 120 degrees and 1.1 atm in four Erlenmeyer flasks.
The previously grown cells were centrifuged at 2500 xg at 4 degrees for 20 minutes, the supernatant was discarded and the pellet was resuspended in 0.6% saline solution.
From this resuspension, the cells were counted in a Neubauer chamber. For this process, we used 100 ul of the cell suspension plus 100 ul of 0.1% methylene blue and counted them using optical microscopy. The counting results are shown in Table 1.
Once this was done, we inoculated 5 ml of this concentrated solution into 50 ml of liquid YDA medium in the 4 previously autoclaved erlenmeyer flasks.
Table 1: Neubauer chamber cell count results for each quadrant
| Quadrant 1 | 134 cells |
| Quadrant 2 | 141 cells |
| Quadrant 3 | 165 cells |
| Quadrant 4 | 160 cells |
In total we had 600 cells, dividing by 4 we have 150 cells
we get 1.5x10^10 cells/ml
AUGUST 21th 2024
The fourth erlenmeyer flasks with the grown cells were centrifuged at 2200 xg for 20 minutes, the supernatant was discarded and the cells were resuspended in water, then centrifuged again under the same conditions and an arbitrary amount of 0.6% saline solution was added to obtain a suspension with a final volume of 6 ml.
To start the weighing process, three porcelain crucibles were carefully washed, dried and numbered from 1 to 3. The crucibles were then placed in an oven at 105°C for 48 hours.
AUGUST 22th 2024
The three crucibles were carefully removed from the oven and kept in the desiccator for 15 minutes. They were then weighed on an analytical balance. After weighing, we put the crucibles back in the oven at 105°C for another hour and in the desiccator for 15 minutes, and weighed them again. As the value of the difference between the two weighings of the crucibles was small, we observed that the moisture content of the crucibles had been minimized as much as possible, making it possible to be more precise with the weighing values. Finally, we added 1.5 ml of the cell solution to each porcelain crucible and left it in the oven at 105°C for 24 hours.
AUGUST 23th 2024
The dried cells were placed in the desiccator for 15 minutes and weighed on an analytical balance. After weighing, we left the cells for another hour in the oven at 105°C and then for 15 minutes in the desiccator, and weighed them again. These weighings enabled us to find the mass of cells in each crucible from the difference between the mass of the empty crucible and the mass of the crucible with dry cells.
Table 2: Weighing of Crucibles
| Empty Crucibles | Crucibles with Dry Biomass | Dry Cell Mass | |||||||
| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | |
| 1st Weighing | 48,5775 | 46,8807 | 49,9244 | 48,6152 | 46,9658 | 50,0104 | 0,0877 | 0,0851 | 0,086 |
| 2nd Weighing | 48,5319 | 46,8815 | 49,9253 | 48,6293 | 46,9834 | 50,0204 | 0,0974 | 0,1019 | 0,0951 |
| Difference Between Weighings | 0,0044 | 0,0008 | 0,0009 | 0,0141 | 0,0176 | 0,01 | 0,0097 | 0,0168 | 0,0091 |
From the initial 6 ml solution we made several dilutions to obtain its absorbance using the spectrophotometer, the results obtained are shown in Table 3:
Table 3: Optical density measurements for different dilutions of the 6 ml solution.
| OD measurement to determine abs | |
| Sample | A600 |
| 1:2000 | 0,11 |
| 1:1500 | 0,27 |
| 1:1200 | 0,25 |
| 1:1000 | 0,22 |
| 1:700 | 0,4 |
| 1:500 | 0,39 |
| 1:200 | 0,99 |
After weighing, we know the mass of cells in each crucible and we know that the volume of cells added to each crucible is 1.5 ml, so we can find the concentration for the different weighing values:
Table 4: Concentration (g/L) in each of the crucibles
| Crucible 1 | Crucible 2 | Crucible 3 | |
| 1st weighing | 58,4667 | 56,7333 | 57,3333 |
| 2nd weighing | 64,9333 | 67,9333 | 63,4 |
| Average | 57,5111 |
With these values, we averaged the concentrations obtained in the first weighing only, obtaining 57.5111 g/L. We only used the first measurements because we felt that they would be the most accurate measurements we obtained during the experiment. We then divided this concentration value by the dilution factor of the mother solution, and thus obtained Table 4:
Table 5: Optical density measurements for different dilutions of the 6 ml solution and their respective concentrations
| OD measurement to determine abs | ||
| Sample | A600 | Concentration (g/L) |
| 1:2000 | 0,11 | 0,02875555556 |
| 1:1500 | 0,27 | 0,03834074074 |
| 1:1200 | 0,25 | 0,04792592593 |
| 1:1000 | 0,22 | 0,05751111111 |
| 1:700 | 0,4 | 0,08215873016 |
| 1:500 | 0,39 | 0,1150222222 |
| 1:200 | 0,99 | 0,2875555556 |
Using these values, we plotted an absorbance X concentration curve (Figure 1) to find the equation that described this behavior
Figure 1: Standard microbial growth curve for the yeast S. cerevisiae SC9721
In this way we obtain a standard microbial growth curve for the yeast S. cerevisiae so that we can determine the concentration of future suspensions that we make during the experiments using the equation described in the graph above.
AUGUST 29th 2024
We started the process of propagating the inoculum for the bioreactor. We used four 250 ml erlenmeyer flasks containing 50 ml of YPD medium in which we inoculated 1 ml of S. cerevisiae grown the previous day (12 hours) in liquid YPD medium. We put the four flasks to grow at 30°C at 180 rpm.
- DETERMINING CELL CONCENTRATION
AUGUST 30th 2024
The culture media with the grown yeasts was centrifuged at 2,200xg for 20 minutes and washed twice with 0.6% saline solution. We then made a 1:1000 dilution of the cell suspension (7.3 ml) and measured its OD using a spectrophotometer, where we obtained an A600:0.26
- BIOREACTOR TEST
AUGUST 31th - SEPTEMBER 09th 2024
Unfortunately due to structural problems in the laboratory it was necessary to stop the experiments for the bioreactor.
SEPTEMBER 17th 2024
We prepared the YDA culture medium containing 100g/L of glucose. This medium was used to grow the yeasts in the bioreactor and prepare the inoculum. This concentration was used because we wanted the bioreactor to have a concentration of 0.1g/L of glucose in order to avoid the Crabtree effect. In total, we made the inoculum using 4 one-liter conical flasks containing 500 ml of medium with 50 ul of cells.
SEPTEMBER 18th 2024
The reactor was prepared with a useful volume of 1 liter containing 0.6% saline solution, 0.1g/L glucose and 3g/L of cells. Aeration was maintained at 0.3L of air/min and PH 5.5 using 4 molar HCL and 4 molar NAOH solutions, while stirring was maintained at 300 rpm.
During the execution of the process, we noticed that the oxygen partial pressure parameter was decreasing a lot, initially we increased the air flow rate to 0.6 L/min and then to 1 L/min, we also changed the agitation to 400 rpm, however, the partial pressure was still decreasing, which suggests that the initial cell concentration was high and therefore we had a large growth of yeast, which increased oxygen consumption.
During the 30-minute interval, the bioreactor was fed with YPD culture medium according to the volumes calculated earlier.
Samples from the bioreactor were taken every 1 hour and centrifuged at 2,200xg for 20 min to separate the pellet from the supernatant. Once this was done, we reserved the supernatant to carry out the DNS test and determine the glucose concentration, while the pellet was resuspended in 1ml of distilled water and then diluted in a 1:10 ratio, then we measured its optical density using a spectrophotometer to find its concentration from its standard growth curve.