Team:Sao Carlos-Brazil/Protocols
Project Protocols
Protocols
Mediums
YM medium
The YM medium consists of 0.3% (w/v) yeast extract, 0.3% malt extract, 0.5% (w/v) peptone, and 1% (w/v) dextrose. The pH was adjusted to 6.5. For solid YM, 2% (w/v) was added bacteriological agar.
YPD medium
The yeast S. cerevisiae was cultivated in YPD medium, composed by 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose. The pH was adjusted to 6.5. For solid YPD, 2% (w/v) bacteriological agar was added.
SC URA- medium
The selective medium SC URA- consists of 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, 0.01% (w/v) leucine, 0.01% (w/v) lysine, 0.01% (w/v) tryptophan, 0.005% (w/v) histidine. The pH was adjusted to 6.5 with NaOH 1 M. For solid SC URA-, 2% (w/v) bacteriological agar was added.
5 FOA selective medium
The selective medium using 5 FOA as a marker consists of a drop-out mixture [0.002% (w/v) adenine, 0.002% (w/v) arginine, 0.002% (w/v) histidine, 0.003% (w/v) leucine, 0.003% (w/v) lysine, 0.002% (w/v) methionine, 0.004% (w/v) phenylalanine, 0.02% (w/v) threonine, 0.002% (w/v) tryptophan and 0.003% (w/v) tyrosine, 2% (w/v) dextrose, 0.77 (w/v) nitrogen base, 0.002% (w/v) uracil, 0.1% (w/v) 5-Fluoroorotic Acid and 2% (w/v) agar.
Yeast transformation using the LiAc/SS carrier DNA/PEG method
Solutions:- Tris EDTA 10x: Tris base 100 mM, EDTA 10 mM pH 7.5.
- Lithium acetate 10x: Lithium acetate 1M pH 7.5.
- PEG 4000 50%: PEG 4000 50% (w/v).
S. cerevisiae transformation protocol
The yeast strain S288C was inoculated in YM liquid medium and incubated overnight on a rotary shaker at 150 rpm, 30 °C. The culture was diluted in 50 mL of YM medium to 0.1 O.D. and incubated at 150 rpm, 30 °C until the culture reached 0.38 O.D. Then the cells were centrifugated at 2,500 rpm for 2 minutes. The medium was discarded and the cells resuspended in 20 mL of water. The mixture was centrifugated at 2,500 rpm for 3 minutes and the supernatant was removed.
The pellet was resuspended in 1 mL 1xTE/LiAc and incubated at 30 °C for 45 minutes to induce competence. The salmon sperm DNA was denatured for 10 minutes at a boiling water bath and then immediately chilled on ice for 2 minutes. After the incubation, the competent cells were divided into negative control (200 uL) and the main reaction (200 uL). In each reaction, 0.12 mg of salmon sperm DNA was added and, in the main reaction, 12 uL of deletion cassette was added. At the tubes, 600 uL PEG 40% was added and the reactions were incubated at 150 rpm, 30 °C for 45 minutes.
After the incubation, 70 uL of DMSO was applied and the cells were incubated at 42 °C for 25 minutes. The cells were immediately chilled on ice for 2 minutes and the tubes centrifugated at 4,000 rpm for 2 minutes. The supernatant was removed and the cells resuspended in 100 uL of water. The cells were plated on selective medium and incubated at 30°C for approximately 2 days.
Transformation by electroporation
The yeast strain S288C was inoculated in 50 mL YM liquid medium and incubated overnight on a rotary shaker at 180 rpm, 30 °C. The culture was centrifugated at 4000g, 4 °C for 2 minutes and the pellet resuspended in 40 mL of water. In the tube, 1 mL of 10xTE and 1 mL of lithium acetate was added. The cells were incubated for 45 minutes at 30 °C, 90 rpm.
After the incubation, 250 uL of DTT was applied and the culture incubated for 15 minutes at 30 °C, 90 rpm. The yeast suspension was diluted to 50 ml with water. The cells were washed three times by centrifuging at 4000 g, resuspending the first pellet in 25 ml ice-cold water, the second pellet in 3 mL ice-cold 1M sorbitol and the third pellet in 0.5 mL ice-cold 1M sorbitol. The equipment used was a Bio-Rad Gene Pulser and the electroporation cuvette was a 0.2-cm-gap. In a sterile, ice-cold 1.5 ml microcentrifuge tube, 40 uL of concentrated yeast cells were mixed with 80 ng transforming DNA.
The liquid was transferred to an ice-cold cuvette and electroporated (pulse at 1.5 kV, 25 uF, 200 Ω). The cuvette was immediately chilled on ice for 2 minutes and 1 mL ice-cold 1M sorbitol was added with gentle mixing using a pipet. The aliquot of yeast suspension was spread directly on selective medium and the plate was incubated at 30 °C for approximately 2 days.
Yeast DNA extraction
DNA extraction Buffer
The DNA extraction buffer consists of 200mM Tris HCl pH8, NaCl 250 mM, EDTA 25 mM, SDS 0.5%.
Procedure
For yeast DNA extraction, 9 reactions were conducted using Jena Bioscience kit (catalog number PP-215XS), gently provided by the company. Some genomic DNA extractions were made using the glass beads method. In this protocol, the cells were inoculated in 10 mL of YPD medium and incubated overnight at 150 rpm, 30 °C.
After the incubation, 1.5 ml of the cell culture were centrifugated at 2.500 g for 5 minutes. The pellet was resuspended in 500 uL of DNA extraction buffer and transferred to an Eppendorf containing glass beads. The tube was vigorously mixed for 10 minutes and the cells lysate was transferred to an Eppendorf containing phenol: chloroform (1:1). The lysate was vigorously mixed for 10 minutes and then centrifugated at 16.000 g for 10 minutes. The supernatant was transferred to a tube containing 200 uL of chloroform and centrifugated at 16.000g for 7 minutes.
The supernatant was transferred to a tube containing 800 uL fo isopropanol and incubated at -80 °C for 30 minutes. The sample was centrifugated for 15 minutes at 16.000 g and the supernatant discarded. The pellet was washed with ethanol 70% and centrifugated for 1 minute at 16.000 g. The pellet was dried for 10 minutes (at the room temperature) and the DNA was eluted in 50 uL of ddH2O.
Stratospheric experimentation protocol: preparation and analysis
Inoculum
100μL of Saccharomyces cerevisiae previously cuLtivated in 2mL of YM medium. It takes 48h to get to the stationary phase.
Cultivation
Cultivation at 2 mL of YM medium at 30ºC in a shaker. The stationary phase is detected when the optic density of a 10-fold dilution is 1,4.
Dishes preparation
Petri dishes without any kind of medium were divided into two sectors. In each of them, twelve dots were added, marking the position where a specific desiccated spot will be placed, and an arrangement of three dots (triplicates of every group) was marked 0, -1, -2 and -3, which refer to the cell dilution used for the desiccation in the next step. One sector was labeled NT (no treatment) and the other M (melanin). The illustration below shows the dishes’ disposition. For every strain, three such dishes were prepared: one for ground samples, one non-exposed stratospheric samples and exposed stratospheric samples.
Desiccation
About two hours before the flight, microtubes with autoclaved double distilled water were used to dilute the stock solution 10, 100 and 1000-fold. Then, 10 μL of each dilution and the stock solution were dropped on the dish at the NT section, directly on the polystyrene. The M section was filled with drops diluted in water mixed with cuttlefish ink (Nortindal). The dishes were then left open in the back of the interior of a laminar flow hood. It usually takes 90 to 120 minutes for desiccation to be complete.
Final flight Preparations
The ground samples were left inside a desiccator. The non-exposed dish (with lid) was covered by Silver Tape (which should be somewhat loose to equalize internal and external pressure) and double-sided bonding tape was laid over the Silver Tape and on the bottom of the dish. The exposed dish was then fixed over the non-exposed one. Afterward, the set of two dishes was fixed to the probe’s laminated carbon fiberboard. It is important to note that all fixation was done using only double-sided bonding tape. Immediately before the flight, the lids of the exposed dishes were removed.
Samples recovery
Recovery around 150 minutes after launch was made by removing the dishes from the carbon fiberboard. This is done by gently twisting the dish set to unstick the bonding tape. The samples were immediately placed inside a plastic recipient and covered with their respective lids.
Samples resuspension and drop plating
Resuspension is conducted by applying 10 μL of 0.9% saline solution to the desiccated spot. The resuspended spot is then placed inside a microtube containing 90 μL of saline solution (10 μL of the resuspended material plus 90 μL for a final of 100 μL). This gives the sample a 10-fold dilution. This procedure was carried out for all desiccated spots, from ground samples to exposed ones. The microtubes were briefly vortexed and each sample was drop plated on YM solid medium. For each sample, three 10 μL drops were placed on YM. This gives a triplicate’s triplicate for CFU counting. The drops must be left in the back of a laminar flow hood to dry out, which takes a few minutes. Afterward, all YM dishes were incubated for two days at 30°C. The initial stock solution used to desiccate the samples was also drop plated as control using -3, -4 and -5 dilutions.
CFU Counting
Sample survival was asserted by visually counting the colony-forming units of every group. There is no need to count all the dilutions, only the one that is the most adequate for the researcher for a specific group. A lab magnifier might be used to best visualize the CFUs.
Data treatment
The simple average of CFU counting for all replicates in a group is calculated. The average is then multiplied by the dilution (which gives N) and this number is then divided by N0 (the control average multiplied by the respective dilution). After this treatment, survival rates of ground desiccated samples (NT and M), non-exposed samples (NT and M) and exposed samples (NT and M) are obtained and a bar graph is plotted using these values (example below). The error bars indicate the standard deviation of the average of N/N0 for triplicates of the respective group. Finally, to ascertain whether the acquired values are statistically significant, a Two-Way ANOVA test was carried out.
