Team:Tartu TUIT/Experiments
While doing scientific experiments, it is important to make and follow uniform protocols to eliminate as many variables as possible and to ensure repeatability of the experiment. We followed protocols provided by both our sponsors and supervisors. If some steps in our experiments failed repeatedly, we changed a few parameters of protocols like incubation time or amount of DNA, however, protocols written here were our main guidelines and we tried not to diverge much far from them.
Phusion DNA polymerase protocol
To amplify DNA fragments for subsequent cloning, PCR with Phusion DNA polymerase was used. Components of PCR reaction are listed in Table 1.
Table 1. Components of PCR reaction(table below)
| Components | 50 µL reaction | Final concentration |
|---|---|---|
| Nuclease-free water | add to 50 µL | - |
| Buffer 5X (HF or GC) | 10 µL | 1X |
| dNTPs mix (25 mM each) | 0.4 µL | 200 µM each |
| DMSO((CH3)2SO) (Optional) | 1.5-2.5 µL | 3-5% |
| Forward primer (10 µM) | 2.5 µL | 0.5 µM |
| Reverse primer (10 µM) | 2.5 µL | 0.5 µM |
| DNA template: Genomic DNA,Plasmid DNA | X µL; X µL | 50 ng - 250 ng; 1 pg–10 ng |
| Phusion DNA Polymerase | 0.5 μLl | 0.02 U/µL |
All the components of the PCR reaction were mixed on ice. PCR tubes with the reaction mix were transferred into a preheated to 98 oC thermocycler block and the PCR were performed according to the conditions listed in the table 2.
Table 2. PCR machine setup(table below)
| Step | Temperature (°C) | Time (s) |
|---|---|---|
| Initial Denaturation | 98 | 30 |
| 25-35 Cycles | 98 | 10 |
| 45-72 (depends on primers’ Tm) | 20 | |
| 72 | 15-30 s per kb | |
| Final Extension | 72 | 15-30 s per kb |
| Hold | 15 | ∞ |
Restriction
All the restriction enzymes used in our work were FastDigest enzymes from Thermo Fisher Scientific. Restriction reactions were prepared by mixing the following reagents (Table 1.):
Table 1. Restriction reaction mix for 20 μL volume(table below)
| Components | Volumes (°C) |
|---|---|
| Water | add to 20 μL |
| 10X FD Buffer | 2 μL (1X) |
| DNA | 1 μg |
| 15-30 s per kb | |
| Restriction enzymes | 1 μL |
| FastAP (for vector DNA only)* | 0.5 μL |
*FastAP (Thermosensitive Alkaline Phosphatase) was added to the vector restriction reaction in order to prevent self-ligation of the vector fragments.
All the components of restriction reactions were mixed in a 1.5 mL tube. After mixing, the restriction reaction was incubated at 37°C for 30-60 min. Following the incubation, restriction enzymes were inactivated by heating the mixture for 15 min at 65-85°C. At the next step, PCR clean-up protocol or gel electrophoresis followed by DNA purification from the gel was performed to clean the restricted DNA fragments.
Agarose gel for DNA separation
1. In order to make 1% agarose gel, 1 g of agarose was added to 100 mL of TAE [tris(hydroxymethyl)aminomethane - ethanoic acid - ethylenediaminetetraacetic acid] buffer solution.
2. The mixture was heated in a microwave until agarose was completely melted.
3. The mix was left to cool down to approximately 60°C.
4. 6 μL of Atlas ClearSight DNA Stain was added.
5. The solution was poured into a gel caster with an appropriate comb installed.
6. After polymerization, the gel was used for DNA electrophoresis.
DNA purification from agarose gel
DNA was purified from the gel using the Gel-Out kit from A&A Biotechnology according to the manual instructions. A brief description of the procedure is written below.
1. DNA fragments of the correct sizes were cut out from the gel and transferred to Eppendorf tubes.
2. R7SI agarose melting solution was added; in case of < 2% agarose gel - 400 μL, in case of ≥ 2% agarose gel - 500 μL. Samples were incubated at 50°C until the complete dissolution of agarose slices. During incubation, the samples were mixed from time to time by inverting the tubes or vortexing.
3. An appropriate volume of Isopropanol was added.
4. < 2% agarose gel - 200 μL.
5. ≥ 2% agarose gel - 250 μL.
6. The samples were mixed by inverting the tubes.
7. After brief centrifugation, the mixture was transferred onto the minicolumns, which were centrifuged for 30 s at 10 000-15 000 RPM.
8. The flow-through was discarded and the minicolumns were re-assembled with the collection tubes.
9. At the next step, 600 μL of A1 wash solution were added and centrifugation for 30 s at 10 000-15 000 RPM was performed.
10. The filtrates were discarded and minicolumns were re-assembled with the collection tubes.
11. 300 μL of A1 wash solution were added.
12. Centrifugation for 1 min at 10 000-15 000 RPM was performed.
13. The dry minicolumns were transferred to the new 1.5 mL elution tubes and 50 μL of TE buffer were added directly onto the minicolumns resin.
14. Minicolumns were incubated for 3 min at room temperature and centrifuged for 60 s at 10 000-15 000 RPM.
15. The minicolumns were discarded. Purified DNA was used right away or kept at -20°C until needed.
DNA concentration measurements
DNA concentration measurements were performed with NanoDrop Microvolume Spectrophotometer according to the manufacturer manuals.
Software used: NanoDrop 1000 v.3.8.1.
Mode used: Nucleis Acid, DNA-50
Ligation
All ligation reactions were performed using T4 DNA ligase from NEB according to the manufacturer's instructions. All the components were mixed in Eppendorf tube in accordance with Table 1. DNA was taken using the 1:3 ratio of vector:insert based on the size of DNA fragments.
Table 1. Components of the ligation reaction (table below)
| Components | Amount |
|---|---|
| Water | add to 20 μL |
| 10X FD Buffer | 2 μL (1X) |
| Vector | 0.020 pmol |
| Insert | 0.060 pmol |
| T4 DNA Ligase | 1 unit (5 units for blunt-end ligation) |
| PEG 4000 (for blunt-end ligation, optional) | 2 μL |
Ligation reactions were kept at 16°C for 2-4 hours or overnight (if ligation contains blunt ends). 1-2 µL of ligation mixture were used for bacterial transformation.
Bacterial transformation
DNA transformation to bacteria cells was performed as follows:
1. Escherichia coli Dh5α we transferred from -80°C freezer to ice. E.coli cells were left on ice for approx. 10 min to melt.
2. 50 μL of cells (DH5α) was mixed with 2 μL of ligation reaction (50 pg - 1 ng for plasmid DNA).
3. Cells were left for 30 min on ice.
4. Heat-shock was performed at 42°C for 30 s.
5. Cells were cooled for 1-2 min on ice.
6. 1 mL of LB medium was added to the cells.
7. Tubes were incubated in the shaker at 37°C and 220 rpm for 40 min.
8. Meanwhile, bacterial selection plates (LB with antibiotics) were transferred from the cold room to room temperature (Plates were labeled accordingly and a few pieces of glass beads were added to each of them).
9. After incubation, Eppendorf tubes with bacteria were centrifuged for 1 min at 5000 rpm.
10. Excess of supernatant was discarded (approx. 250 µL were left in the tube).
11. Bacterial cells were resuspended in the supernatant leftover and transferred on the selection plate.
12. Bacterial cells were spread on the plate surface evenly using sterile glass beads.
13. Glass beads were removed and plates were incubated at 37°C for 12-14 hrs (until single colonies appeared).
Plasmid extraction
Plasmids were extracted from bacterial cells using the “Plasmid Mini” kit by A&A Biotechnology according to manual instructions. A brief description of the procedure is written below.
1. Up to 3 mL (1.5-3 mL) of overnight bacterial culture was centrifuged. After that supernatant was removed and the bacterial pellets were suspended in 200 µL of L1 cell suspension solution.
2. 200 µL of L2 lysis solution was added and the solution was carefully mixed by turning the Eppendorf tube up-side-down. The mix was incubated for 3 min at room temperature.
3. 400 µL of GL3 neutralizing solution was added and carefully mixed until the disappearance of the raspberry color of the lysates.
4. The lysates were centrifuged for 10 min at 10 000-15 000 RPM.
5. The supernatants were transferred onto the minicolumns, which were centrifuged for 1 min at 10 000-15 000 RPM.
6. The minicolumns were removed from the collection tubes and the filtrates were discarded then the minicolumns were re-assembled with the same collection tubes.
7. 500 µL of W first wash solution were added and the minicolumns were centrifuged for 1 min at 10 000-15 000 RPM.
8. The minicolumns were removed from the collection tubes, the filtrates were discarded and the minicolumns re-assembled with the same collection tubes.
9. 600 µL of A1 second wash solution were added and centrifugation for 2 min at 10 000-15 000 RPM was performed.
10. The dry minicolumns were carefully transferred into new 1.5 mL tubes. 60 µL of TE buffer was added directly onto the minicolumns resin to elute DNA.
11. Minicolumns were incubated for 3 min at room temperature followed by 1 min centrifugation at 12 000 RPM.
12. Eluted plasmid DNA was used straight away or stored at -20°C until needed.
Yeast transformation
In order to genetically transform yeast cells, the desired yeast strain was pre-grown in 50 mL of liquid YPD (yeast extract peptone dextrose) until OD600 0.6-0.8. The genetic transformation was performed based on the following protocol:
1. Cell culture was centrifuged in a sterile 50 mL falcon tube. The supernatant was discarded.
2. The cell pellet was resuspended in 1 mL of sterile 100 mM LiAc(Lithium Acetate) in TE (tris(hydroxymethyl)aminomethane - Ethylenediaminetetraacetic acid buffer) and transferred into the Eppendorf tube.
3. Cells were centrifuged at 3600 rpm for 60 s. The supernatant was removed and cells were resuspended again in two times of cell volume of 100mM LiAc in TE.
4. Cells were incubated for 10 min at room temperature (competent cells).
5. Single-stranded carrier DNA was prepared: Salmon Sperm DNA(SS-DNA) was boiled for 10 min at 100°C and immediately chilled on ice before use. This ensured that the DNA was single-stranded. SS-DNA was kept on ice for the rest of the time before use.
6. 20 μL of plasmid DNA (yeast integration vector restricted with proper restriction enzyme) and 10 μL of SS-DNA was mixed in a separate 1.5 mL tube.
7. 100 μL of yeast competent cells were added to the DNA and mixed.
8. At the next step, 700 ul of sterile PEG(Poly(ethylene glycol))/LiAc (40% PEG 3350 + 100mM LiAc in 1 x TE) were added.
9. Finally, 48 μL of DMSO((CH3)2SO) was added and everything was gently mixed by pipetting.
10. The mixture was incubated at 42°C for 40 min (heat shock).
11. After the heat shock, the cells were chilled on ice for 2 min.
12. At the next step, the cells were centrifuged for 30 s at 6000 rpm at room temperature. The supernatant was removed and the cells were resuspended in 1 mL of sterile 1x TE buffer.
13. After that, the cells were centrifuged for 60 s at 3600 rpm at room temperature. The supernatant was discarded and the cells were resuspended in 200 μL of sterile 1x TE buffer.
14. Finally, 200 μL of suspension were transferred on the yeast selection plates and cells were evenly spread all over the plate surface using sterile glass beads.
15. The plates were incubated upside down at 30°C until colonies appeared (usually, for 2-3 days).
Yeast colony PCR
Preparation of yeast cell lysates
1. Cells of the single yeast colony (at the end of the pipette tip) were transferred into 30 µL of 20 mM NaOH in the Eppendorf tube.
2. The mixture was boiled at 100°C for 10 min.
3. Lysates were placed on ice for cooling followed by 1 min centrifugation at 12000 rpm.
4. Yeast cell lysates were placed on ice.
5. 1 µL of supernatant was used as a template for PCR reaction.
Preparation of PCR mixture
Components of the PCR reaction were mixed according to Table 1.
Table 1. Components for PCR with Taq polymerase(table below)
| Components | 20 μL Reaction |
|---|---|
| Water | till 20 μL |
| dNTPs (25 mM each) | 0.25 μL |
| Primers (10 μM) | 0.5 μL each |
| MgCl2 (50 mM) | 1 μL |
| 10xYPB Buffer (Solis BioDyne) | 2 μL |
| Taq polymerase (Solis BioDyne) | 0.3 μL |
| DNA template (cell lysate) | 1 μL |
Conditions used for yeast colony PCR are described in Table 2.
Table 2. PCR machine setup(table below)
| Step | Temp | Time |
|---|---|---|
| Initial Denaturation | 95o C | 5 min |
| 25-35 Cycles | 98 (Denaturation) | 30 s |
| 48 o - 60 o C (Primer annealing) | 20-35 s | |
| 72 o C (Elongation) | 1 min/kb | |
| Final Extension | 72o C | 5 min |
| Hold | 15o C |
LB(Lysogeny broth) and YPD media
Table 1. LB medium components(table below)
| Tryptone | 10 g |
| Yeast extract | 5 g |
| NaCl | 10 g |
| H2O up to | 1 L |
Table 2. YPD components(table below)
| Peptone(Formedium) | 20 g/L |
| D-Glucose anhydrous | 20 g/L |
| Micro Granulated Yeast Extract | 10 g/L |
| Bacto agar(for plates) | 15 g/L |
1. All the components were mixed in 2 L Erlenmeyer flask with a magnetic stirrer in it.
2. 15 g of Bacto Agar (for plates only, Bacto Agar was omitted for liquid medium) were added.
3. The medium was autoclaved for 15 min at 121°C.
4. The medium was left to cool down to approximately 55°C.
5. 1 mL of sterile 100 mg/mL antibiotic solution (penicillin) was added to the medium and mixed.
6. Approx. 30 ml of medium was poured into the sterile Petri dishes and was left for cooling down and polymerization.
7. Plates were kept at +4°C for up to two weeks.
Microscopy
For the promoter characterization part, yeast colonies were checked after the transformation for the presence of GFP fluorescence. Prior to the experiment, yeast cultures were grown in CSM uracil dropout medium with 2% glucose as a carbon source. The optical density (OD600) was in the range 0.2-0.6. Cultures were observed under a Zeiss Axio Observer Z1 microscope. The following settings were used for the microscope: 25 ms exposure time for phase contrast channel, 100 ms exposure time for EGFP channel. Colibri LED modules were used as a source of 470 nm excitation wavelength at 25% intensity.
Time-lapse microscopy
Prior to time-lapse microscopy, the yeast cells were grown in synthetic complete (SC) media lacking histidine or SC lacking histidine and tryptophan depending on the integrative plasmids in the strain. The cultures were grown to OD600 0.2-0.8, the cells were pipetted onto a 0.08 mm cover glass slip and were covered with 1.5% agar-SC. For induction of the estradiol-controlled promoter, 1 μM estradiol was added to the SC-agar. Zeiss Observer Z1 microscope with 63C/1.4NA oil immersion objective and Axiocam 506 mono camera was used for imaging. The focus was kept using Definite Focus and the sample was kept at 30 °C using PeCon TempControl 37-2 digital. The cells were imaged every 5 minutes using brightfield illumination and the experiments were 13-18 hours long.
Plate reader
Plate readers are instruments that are used to detect samples in microtiter plates. This year as a team has used BioTek Synergy MX microplate reader to measure the cells’ optical density and intensity of EGFP fluorescence. All strains used in the experiments were pre-grown overnight at 30°C in liquid CSM/2%Glc media and diluted to OD600 in a range of 0.9 to 1.1. Then diluted cell cultures were distributed to 96 well plates (clear flat bottom).4 replicates of 2 colonies from each strain were used. 200 µL of cells were added to each well. CSM/2%Glc media was used as a reference to OD600. The measurements were done with the following parameters. Absorbance was measured at 600 nm and when it comes to fluorescence it was measured at these conditions -excitation 485 nm, emission 528 nm, bandwidth 20, gain 80.
Western blotting
1. The yeast strains were pre-grown in 10ml CSM media overnight in the 300C shaker.
2. Before the induction of Glucanase expression (Timepoint 0), 5ml of cell culture was collected (centrifugation at 4000 rpm for 1 minute), proteins in the supernatant were concentrated using Acetone precipitation method.
3. Another half of the cell culture was induced with Beta-estradiol (final concentration 1μM) and incubated for 6 hours.
4. After 6 hours incubation, cells and supernatant were collected as described in 2.
5. Cell pellets were resuspended in 200 µl of Urea buffer, 250 µl of glass beads were added.
6. Cells were disrupted using BeadBeater(4 m/s for 40s)
7. The samples were centrifuged for 10 min at 14200 rpm.
8. Protein precipitates from the supernatant were resuspended in 200 µl of Urea buffer.
9. Samples were loaded to the SDS-PAGE.
10. SDS-PAGE (Denaturing Poly acrylamide gel electrophoresis) was performed using the following parameters: 15 mA, 300V 1h 20 min.
11. To transfer proteins from the gel onto a membrane the semi-dry western blot transfer method was used: 60 mins transfer at 25V and 1A.
12. The nitrocellulose membrane has high affinity for proteins and antibodies and therefore its necessary to minimize the nonspecific antibody binding. The membrane is blocked in 5% dry milk, 1X TBS-T buffer solution, and incubated with agitation for 1 hour.
13. After blocking, the membrane is then incubated with agitation for 1 hour in solution containing blocking solution and 1:1500 dilution of primary antibody (Anti-HA) that specifically recognizes protein of interest.
14. This is followed by washing with 1X TBS-T buffer for 15 mins and 3 more times for 5 mins with fresh buffer under mild agitation to remove nonspecific binding primary antibody.
15. The membrane was then incubated with secondary antibody (1:7500 dilution rabbit anti-mouse in 3% dry milk, 1X TBS-T buffer solution) with agitation for 30 mins and then washed with 1X TBS-T buffer to remove excess secondary antibody: 1 time for 15 minutes and 3 times for 5 minutes in quick stirs.
16. Result manifestation using Supersignal West Pico Rabbit IgG Detection Kit. Exposure time used ranged from 10 seconds to 10 minutes.
Concentration of the proteins from the supernatant
Acetone precipitation of proteins
Materials Required
• Cold (-20°C) acetone, a volume four times that of the protein samples to be precipitated
• Centrifuge tube, made of acetone-compatible polypropylene and able to hold five times the sample volume
• Centrifuge and rotor for the tubes used, minimum 13,000 × g required
Protocol
1. Cool the required volume of acetone to -20°C.
2. Place the protein sample in an acetone-compatible tube.
3. Add four times the sample volume of cold (-20°C) acetone to the tube.
4. Vortex tube and incubate for 60 minutes at -20°C.
5. Centrifuge 10 minutes at 13,000-15,000 × g.
6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet. Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat steps 2-5 before proceeding to step 7.
7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it may not dissolve properly.
8. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve the protein pellet.
Acetone precipitation of proteins
Amicon Ultra-15 Centrifugal filters
1. Add up to 15 mL of sample (12 mL if using a fixed-angle rotor) to the Amicon® Ultra filter device.
2. Place capped filter device into centrifuge rotor; counterbalance with a similar device.
3. When using a swinging-bucket rotor, spin the device at 4,000 × g maximum for approximately 15–60 minutes. When using a fixed-angle rotor, orient the device with the membrane panel facing up and spin at 5,000 × g maximum for approximately 15–60 minutes.
4. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.
NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation.
Amicon Ultra-15 Centrifugal filters
HPLC
Column used: BioRad Aminex HPX-87 H.
Mobile phase - 0.005M H2SO4.
Flask preparation:
1. Grow S.cerevisiae W303 wild-type strain in 5 ml YPD in 15ml tube overnight.
2. Change the media to 5ml 1% glucose minimal Delft medium. Keep cells for 6 hours.
3. To prepare 125ml flask with 20ml of 1% glucose minimal Delft medium and culture with initial OD600 ~0.1. Make triplicates.
4. Check OD600 and collect 500 µl of culture for the HPLC sample every 2 hours during the exponential phase and take one point in 24 hours.
Sample preparation for HPLC:
1. Centrifuge collected samples with 5500rm for 5 min.
2. Transfer the supernatant to another Eppendorf tube.
3.Centrifuge with maximum speed for 5 min.
4. Transfer the supernatant to another Eppendorf tube.
5. Prepare glass tubes with 200 µl of the sample.
6. Run each sample three times.
Spot viability assay
1. Cells were grown for 12 hours at 30°C 160 rpm with and without the induction. 1 µM Estradiol was used.
2. Cultures were diluted to the concentration of 10^6 cells/ml (0.08 OD) in YPD media, YPD with 10% DMSO, to make membrane permeable, and in water, to create hypo-osmotic stress conditions.
3. Serial dilutions were made(1, ¼, 1/20, 1/200)
4. 5 µl of the dilutions were plated on the YPD plates.
5. Cells were grown for 24 hours at 30°C.