Team:DTU-Denmark/Experiments

Experiments

Synthlab protocols

DNA from IDT will typically be delivered in a white flaky substance, which need to be resuspended, before the DNA is ready for use

Materials

Table centrifuge
EB/TE buffer
Genes/Primers from IDT or other DNA provider

Procedure

Quickly spin the DNA down in the table centrifuge
Calculate the amount of EB buffer need to dilute the gblocks to a desired concentration. Important: gene fragments and primeres are not diluted to the same concentration: The concentration of gene fragments is usually 25 ng/μL and for primers it is 100 μM.
Genes/Primers from IDT or other DNA provider
- DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is 40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples)
- In order to calculate the the molar amount of primer, use the NEB calculator
Add the calculated amount of EB buffer
Store the resuspended DNA at -20°C

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Mycolab protocols

Basic protocol on plating of the fungi.

Materials

Agar plates with media of interest
Sterile toothpicks

Procedure

Plating using mycelia

Open the plate containing the mycelia from the species of interest
With a sterile toothpick, scratch the surface of the mycelia.
Spike the new agar plate with the toothpick.
Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.
Put the plate into a plastic growth bag.
Place growing bag into the incubator.

Plating using spores

Touch the sterile tooth pick in the spore suspension.
Spike the new agar plate with the toothpick.
Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.
Put the plate into a plastic growth bag.
Place growing bag into the incubator.

Minimal media used for protoplastation of A. oryzae. This is for 1 L of media.

Materials

50 mL D-glucose
50 mL Nitrate salts
1 mL Trace elements
1 mL Thiamine
20 g Agar (SO.BI.GEL)

Procedure

Mix

Mix in a blue cap flask.
Add water until the volume is 1 L.
Autoclave.

Minimal medium: Schizophyllum commune SMM agar (1L) needed for S.commune plates

Materials

Glucose - 20g
Monopotassium phosphate KH₂PO₄ - 0.46g
Potassium phosphate dibasic K₂HPO₄ 3H₂O - 1.28g
Magnesium sulfate MgSO₄ 7H₂O - 0.5g
Trace elements solution - 1ml
FeCl₃ solution - 1ml
L- Asparagin - 1.5g
Agar - 20g
Thiamine (10mg/100ml) - 1.2ml

Procedure

Preparation of the media

Weigh and add the required solid components (except agar)
Add the required liquid components in a bottle
Dissolve in demineralized water until the total volume is 900ml
Stir the liquid using a magnet (no pH adjustment is required)
Add the required quantity of agar
Add demineralized water until the desired total volume (1L)
Autoclave
After sterilization add 1.2 ml of filter sterilized thiamine

Protocol provided by Fabiano Jares from DTU Bioengineering. The protocol is for species of Aspergillus. We used it for Aspergillus oryzae.

Materials

Aspergillus protoplastation buffer (APB) 1L solution

Final conc: 1.1 M MgSO₄ and 10 mM Na-phosphate buffer. (Hint: use 1 M solutions of Na₂HPO₄ and NaH₂PO₄ to prepare the sodium phosphate buffer). pH is adjusted with 2 N NaOH to 5.8.

APB with Glucanex (40 mg Glucanex/ml APB)
Aspergillus transformation buffer (ATB) 1 L solution

Final conc: 1.2 M Sorbitol; 50 mM CaCl₂·2 H₂O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.

PCT (200 mL stock solution)

Final conc: 1.2 M Sorbitol; 50 mM CaCl₂·2 H₂O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.
Final conc: 50 % w/vol PEG 8000; 50 mM CaCl₂; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store PCT at 4 °C.

Procedure

Protoplastation

Collect the conidia from a plate by adding 5 mL of sterile liquid MM (or YPD for A. niger) with required supplements and firmly rubbing the colonies with a sterile Drigalski spatula. The conidial suspension is withdrawn from the plate and added to the shake flask containing 100 mL of media.
The culture is incubated over night (or 2 days for A. niger and other strains growing slower) at appropriate temperature and 150 rpm.
Harvest the mycelia and germlings by using Mira cloth.
Wash the mycelia with Aspergillus protoplastation buffer (APB) to remove the liquid media from the mycelia.
Resuspend the mycelium in 10-20 ml APB solution containing 40 mg Glucanex/ml APB. Homogenize mycelial and enzyme suspension gently to obtain the best possible digestion of the fungal cell wall.
Shake at the 30°C and 150 rpm for 2-3 hours.
Filter through Mira cloth and collect the flow through.
Add APB up to the total volume 40 ml.
Carefully make an overlay with 5 ml of 2 fold diluted Aspergillus transformation buffer (ATB). Dilute with sterile Milli-Q H2O.
Centrifuge at 3000xg (acceleration 9, deceleration 4) for 12 min.
Upon a successful protoplastation, a halo of white protoplast slurry is caught just below the surface. Collect protoplast slurry and transfer it to new tube.
Add ATB up to the total volume 40 ml.
Centrifuge at 3000xg (acceleration 9, deceleration 9) for 12 min. Discard supernatant.
Resuspend the protoplasts in approx.. 1 ml ATB.

Transformation

Gently mix DNA and protoplast in an eppendorf tube (for a simple transformation 50μL protoplast is enough).
Add 150 μL PCT.
Mix by inversion of the tube.
Incubate 10 min at room temperature.
Add 250 μL ATB and mix.
Plate on osmotic stabilized, selective media.

Notes

The volume of DNA in water should preferably be kept below 25 % of the total volume of DNA-protoplast mix to avoid osmotic stress in protoplasts. The amount of DNA needed for a successful transformation varies with the type of the DNA substrate.

For linear DNA, add ≈10 μL linearized plasmid
Use self-replicating plasmids as positive controls to test the competence of the protoplasts and evaluate the success of transformation. AMA1 plasmids (pLAT4-3): add ≈2 μL miniprep.
Remember to include a negative control plate, where protoplasts are plated without any DNA.

Protocol for protoplastation of Ganoderma resinaceum provided by Ecovative Designs. This is the original protocol that we received from Ecovative. Modifications have been made every single time, it has been used due to limitations in our own lab.

Materials

Glucanex (Sigma L1412)
Driselase (Sigma D9515)
Potato Dextrose Agar (pre-mix) + 5g/L Bacteriological Agar
Potato Dextrose Broth (pre-mix)
Sterile Millipore Water
Osmotic Buffer (500mL)

74.04g MgSO₄ (0.6M)
1.05g MOPS (10mM)
Bring volume to 500mL with MilliQ water
Autoclave

Sorbitol Solution (500mL)

91.1g D-sorbitol (1M)
1.05g MOPS (10mM)
Bring volume to 500mL with Millipore water
Autoclave

Lysing Solution (10mL)

Add 100mg of Driselase to 5mL osmotic buffer in a 50mL centrifuge tube.
Allow to dissolve for 15-20min at room temperature with gentle swirling.
Add 100mg of Lysing Enzyme from Trichoderma harzianum (Glucanex) to 5mL osmotic buffer in a 50mL centrifuge tube.
Allow to dissolve for 15-20min at room temperature with gentle swirling.
Centrifuge the Driselase solution at 10,000xg for 1min to pellet the starch component.
Pour the supernatant into the tube with the Glucanex solution.
Attach a 30mm diameter 0.22um syringe filter to a 5mL syringe.
Fill the syringe with the lysing solution and filter into a 15mL centrifuge tube.
Pour the rest of the lysing solution into the syringe and filter.
Final, sterile lysing solution is 10mg/mL Driselase and 10mg/mL Glucanex in osmotic buffer.

Procedure

Tissue Generation

Inoculate potato dextrose agar plates with a single piece of tissue from a stock plate.
Parafilm and incubate at 30°C for 5 days.
Scrape tissue from the agar surface and place into 1mL of potato dextrose broth in a microcentrifuge tube.
Avoid bringing any agar into the tube.
Vortex the tubes at full speed for 30-60sec.
Place tubes in a tube rack and secure with tape.
Place the tube rack in a shaker incubator so that the tubes are completely horizontal.
Incubate at 30°C and 85rpm for 24hrs

Enzymatic digest

Centrifuge tubes at 10,000xrcf for 5min.
Gently discard supernatant.
Re-suspend tissue pellet in 1mL sterile water.
Invert tube several times and vortex briefly. Do not vortex too long or mycelium will begin to shear.
Centrifuge tubes at 10,000xrcf for 5min.
Gently discard supernatant.
Re-suspend tissue pellet in 1mL osmotic buffer.
Invert tube several times and vortex briefly.
Centrifuge at 10,000xrcf for 5min.
Gently discard supernatant.
Re-suspend tissue pellet in 1mL osmotic buffer.
Invert tube several times and vortex briefly.
Centrifuge at 10,000xrcf for 5min.
Gently discard supernatant.
Re-suspend tissue pellet in 1mL lysing solution.
Invert tube several times, but do NOT vortex.
Place tubes in a tube rack and secure with tape.
Place the tube rack in a shaker incubator so that the tubes are completely horizontal.
Incubate at 30°C and 85rpm for 19-24hrs.

Isolation of Protoplasts

Invert tube several times and vortex to detach and protoplasts from the central cell mass.
Place a 40um cell strainer in a new 50mL centrifuge tube.
Use a 1000uL pipette to further suspend the lysate. Pipette the lysate into the cell strainer and allow to drain fully.
Pipette 1mL of the sorbitol solution into the microcentrifuge tube used in the digestion and invert for 1min to rinse any additional protoplasts.
Pipette this volume through the same cell strainer and allow to drain fully. Cell strainer can be gently spun to remove more of the solution.
Repeat steps 3-5 for remaining protoplast preps (up to 5 additional 1mL preps).
Pour an additional 6-10mL of the sorbitol solution into the cell strainer and allow to fully drain.
Remove the cell strainer from the centrifuge tube.
Gently invert the tube 5-10 times to thoroughly mix the solutions.
Place the tube in a tube rack at 4C and incubate for at least 24hrs.
Centrifuge at 10,000xg for 10min.
Carefully pour the supernatant into a new 50mL centrifuge tube. Try to avoid disturbing the pellet along the side and bottom of the tube. The supernatant can be stored at 4C and more protoplasts may be able to be isolated at a later date, if needed.
Re-suspend the pellet into 1mL of sorbitol solution and transfer to a new 1.5mL microcentrifuge tube.
Prepare a 1:10 dilution in sorbitol solution and count protoplasts via hemocytometer.
Store protoplast suspension at 4°C.

Modifications and notes

There were no Driselase in our lab, so we did the protocol with three different concentrations of Glucanex: 20 mg/ml, 30 mg/ml and 40 mg/ml for the lysing solution after consulting Ecovative.
Second time we tried combining the protoplastation protocol we already had for A. oryzae. We used different conditions in three places with all in all 16 samples: 8 of them were incubated with a shaking speed of 85 rpm and 8 at a speed of 150 rpm. Of these 8, 4 of them were incubated for 2-3 hours and the 4 others for 19-24 hours. Each of the four samples had a different concentration of Glucanex for the digestion: 10 mg/ml, 20 mg/ml, 30 mg/ml and 40 mg/ml.
As there were no cell strainers in the lab, autoclaved funnels with mira cloth were used instead.
The third time we worked with 32 samples instead of 16. The conditions were still the same as the previous run but for increased amount of mycelium we ran 2 two samples for each unique combinition of conditions

To obtain the spores generated from a plate culture in agar.

Materials

MilliQ (MQ) water
Mira-cloth
Autoclaved Funnels
Falcon tubes (50 ml)
Cell spreader

Procedure

Spore suspension

Pour MQ water into the fungal culture with plates.
Rub the plate with the spreader. Make sure the spores get into solution. Spores are hydrophobic and can spread easily into the surroundings.
After spores are in suspension, if more plates with the same spores are to be harvested, pour water and spores to the next plate. Repeat steps 1-3 for as many plates you have.
Set a filter (funnel + miracloth) on a Falcon tube.
Pour MQ water + spores onto the miracloth and filter solution. Make sure all liquid goes through the filter.
Do a second filtration on a new Falcon tube and filter. Make sure all liquid goes through.

Spore counting

Make 1:100 dilution with the spore suspension (only 10 microL needed).
With a counting chamber under the opticalmicroscop, put 5 µL of the dilution into the center of the chamber. Count spores in one of the squared cells.
Calculate spore concentration.