Protocol 2: Culturing Spizellomyces punctatus (Sp) prior to transformation day

Abstract

In order to increase transformation efficiency, a more synchronous culture of Sp is needed. This process involves subculturing active Sp plates in the days leading up to transformation day. At least two generations of Sp should be grown on antibiotic free K1 medium to ensure no trace antibiotics are present in the culture for transformation day. Proper sterile technique should be followed at all times when maintaining cultures of Sp; use either a laminar flow hood or work in the sterile area around an open flame.

Before start

In order to prepare enough plates to provide an adequate amount of zoospores for transformation, subculture one plate of Sp per plasmid to be transformed. One active plate is enough to subculture 2-3 new plates. Complete this protocol roughly 36 hours and then again roughly 18 hours prior to the intended transformation time.

Attachments

Spizellomyces_transformation_steps.pdf

Steps

Flood enough active Sp plates each with 1mL of DS to fit your needs.

Note

One active plate is enough to seed 2-3 new plates and you will need one plate of active Sp per plasmid to be transformed on transformation day.

Holding the plate at an angle, gently wash the DS over the agar.

2.1.

Wash the 1mL of DS over the agar. (1/3)

2.2.

wash the 1mL of DS over the agar. (2/3)

2.3.

Wash the 1mL of DS over the agar. (3/3)

Collect all zoospores into a 50 mL conical.

Filter all zoospores into a new 50 mL conical using the sterile 25 mm syringe filter preloaded with Whatman Grade 1 filter paper.

Inoculate the proper amount of new K1 plates for your needs with 250µL to 500µL of filtered zoospores.

Note

The amount of spores transferred to new plates depends on the density of the plate being harvested, be careful not to overcrowd the plates, as zoospore release will decrease.

Add 500µL of fresh DS to the newly inoculated plates.

Gently tilt the plates to spread the liquid across the entire surface of the agar.

Incubate at 30°C in a humidity chamber.

Note

Using a humidity chamber is standard practice, as it prevents the agar from drying out. Placing the plates into a plastic bin with a beaker of ~20mL of water should be enough.Complete this protocol in its entirety 36 and 18 hours prior to intended transformation time.

Abstract

Before start

Attachments

Steps

Steps

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