Protocol 5: Agrobacterium-mediated transformation of Spizellomyces punctatus (Sp)

Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

Published: 2022-12-28 DOI: 10.17504/protocols.io.n92ldpp5xl5b/v1

Abstract

Once you have completed protocols 1-4, you are ready for transformation. This protocol is lengthy, requires accurate timing, and takes a minimum of 4 days to see results. Zoospores will need to be harvested from the plates prepared in Protocol 2; the process of harvesting is described below. Once Agro is at the appropriate OD, transformation is carried out by co-culturing Agro and Sp on induction media (“IM”) to induce the expression of Agro’s virulence genes and transformation of Sp cells. Co-culturing must be done on IM plates with premade depressions in the agar (see Protocol 4: Creating depressions in induction media plates).

Before start

On transformation day, Agro must be diluted to an OD₆₆₀ of 0.15 and then grown to an OD₆₆₀ of 0.6. Under our laboratory conditions, this takes about 4-6 hours, but this time should be empirically tested for each lab before starting to ensure proper and repeatable results. Be sure to take this growth time into account when planning all steps on and prior to transformation day.

Attachments

Spizellomyces_transformation_steps.pdf

Steps

Growing Agro to the appropriate OD660

Dilute each overnight Agro culture to an OD₆₆₀ = 0.15 using IM.

Note

In our experience, this works out to be 500µL of overnight culture that has been spun for 4500rcf,undefine,0h 5m 0s and then resuspended into 4mL of IM. Dilution conditions should be empirically tested for each laboratory.

Grow the Agro at 28°C and shaking at 225rpm until OD₆₆₀ = 0.6.

Note

Under our laboratory conditions, this takes about 4-6 hours. Growth time to reach optimal OD₆₆₀ should be empirically tested for each laboratory.

Harvesting Sp zoospores for transformation

Flood all wild type Sp plates with 1mL of DS or IM for 1h 0m 0s.

Note

Do this about one hour before Agro has reached an OD₆₆₀ of 0.6.The solution used should have little to no effect on transformation success.

Harvest Sp zoospores by running 1mL of fresh DS or IM along the agar of one plate, holding the plate at a 45 degree angle.

Note

Do this 0h 20m 0s before Agro has reached an OD₆₆₀ of 0.6.

Take the liquid from the first plate and use it to harvest the zoospores on a second plate in the same manner.

Do this for all plates of Sp that were flooded in step 3.

Note

If the volume becomes too much to hold in the serological pipette, empty the contents into a 50 mL conical tube (or other appropriately sized tube), then use 1mL of this zoospore suspension to continue harvesting the remaining plates.

Pool all zoospores into a 50 mL conical (or other appropriately sized tube).

Pass the zoospore suspension through a 40 μm mesh filter into a new 50 mL conical tube.

Note

This removes clumps of cells that could clog a syringe filter.

Use a 18 gauge needle and an appropriately sized syringe to take up all of the 40 μm-filtered zoospore suspension.

Note

Be very gentle, the needle may damage the cells. Alternativel y: Directly load a 25 mL syringe with the 40 μm-filtered zoospore suspension by using a p1000 set to 1000µL and gently pushing the cells into the syringe through the tip.

10.

Use a sterile 25 mm syringe filter preloaded with grade 1 Whatman paper to further filter the spores into a new 50 mL conical tube (or other appropriately sized tube).

Note

PRESS SLOWLY, or risk damaging spores. As a rule of thumb, do not filter more than 5 mL of zoospores for one syringe filter. This suspension is now to be used in co-culturing and should have a concentration of zoospores between 1-10 x10⁶ cells/mL.

Co-culturing Agro and Sp in varying ratios

11.

Per each control and plasmid to be transformed, prepare and label four microcentrifuge tubes according to Figure 3.

12.

Pipette the appropriate amounts of IM and the final solutions from protocols section "Growing Agro to the appropriate OD₆₆₀" and "Harvesting Sp zoospores for transformation" into the appropriate microcentrifuge tube in the order they appear in Figure 3.

Note

This will prevent cross contamination between strains. Pipetting time can be sped up by using an M4 repeat pipettor (Eppendorf #4982000322) to dispense 50µL volumes at a time, ensuring the proper amount is dispensed and that the liquid does not splash due to the force of dispensing. This is best done by dispensing along the tube walls just above the water line.

13.

Gently pipette to mix the contents of the microfuge tube.

14.

Transfer all 200µL of an Agro-Sp co-culture to one of the premade depressions on a room temperature IM plate.

Note

All 4 co-culture tubes for one plasmid can be placed onto one IM plate. One tube per quadrant. Start on the left side and then go to the right side to lower the risk of contamination (or the opposite for left handed people).

15.

GENTLY slide the IM plates to a place where they will not be disturbed.

Note

Avoid picking up the plates, as this may result in the pools of co-culture tocombine, disturbing the ratios established and potentially lowering transformationefficiency or success.

16.

Leave the plates to dry at Room temperature for 12-24 hours.

17.

Seal plates with parafilm, invert, and grow in a closed chamber (such as a plastic storage container) at Room temperature for 4 days.

Note

Evidence of growth appears as an opaque, roughly circular area on the agar. If not all of the quadrants have obvious growth, that is okay. As long as there is at least one quadrant that shows growth, Protocol 6: Selecting for Spizellomyces punctatus transformants can be done, though success at gaining selectable transformants may be lower.

Protocol 5: Agrobacterium-mediated transformation of Spizellomyces punctatus (Sp)

Abstract

Before start

Attachments

Steps

Growing Agro to the appropriate OD660

Harvesting Sp zoospores for transformation

Co-culturing Agro and Sp in varying ratios

推荐阅读