TITLE: Protocol 1: Electroporation of Agrobacterium tumefaciens with a plasmid of interest
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
Electroporation is a widespread method of transforming competent Agrobacterium tumefaciens (Agro) cells with a plasmid containing a T-DNA of interest. The resulting Agro can be used to transform various plants and fungi, resulting in transformed cell lines. This protocol outlines the standard electroporation protocol we use to transform Agro in preparation for Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.

[BEFORE_START]
Electroporation should occur at least 4 days prior to the intended Spizellomyces transformation time to ensure that active, single colonies are available to be selected and transferred to liquid culture the night before transformation day (see Protocol "Growing liquid cultures of Agrobacterium prior to transformation day").

[GUIDELINES]
Any binary plasmid that works in Agrobacterium tumefaciens strain EHA105 (GoldBio #CC-225-5x50) can be used for this procedure. We have had success using plasmids derived from pPZP201-BK (PMID: 7919218). It is imperative that all steps be carried out at 4℃ up until electroporation. Ensure proper sterile technique throughout this protocol; perform all steps except centrifugation and electroporation charge delivery in a laminar flow hood or in the sterile area around an open flame.

[STEPS]
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1. Cool the following materials on ice at least 20 min prior to starting:

Plate with a lawn of wild-type Agrobacterium grown overnight at 28 °C. 
Purified plasmid(s) of interest.
Molecular biology grade water, sterile.
10% (v/v) glycerol, sterile.
1.5 mL centrifuge tube(s), enough to hold the volume of Agro harvested.
0.5 mL centrifuge tube(s), one per plasmid to be transformed, plus controls.
2 mm electroporation cuvettes, one per plasmid to be transformed, plus controls.

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2. Add 1 mL to 2 mL of ice-cold water to the plate of Agrobacterium. Hold the plate at ~45 degrees and run the water over the surface at least 3 times, gently scraping along the agar if necessary to recover the lawn of bacteria. 
 


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3. Transfer the 1 mL of harvested cells to a 1.5 mL centrifuge tube, immediately place back on ice.

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4. Pellet the cells at 4000 rcf, 5 min in a rotor prechilled to 4 °C. 
 

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5. Remove the supernatant and gently resuspend the cells in 1 mL of water. Do not vortex. 
 

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6. Repeat steps 4 and 5, 2 more times for a total of 3 washes.
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7. Remove water and resuspend the cells in 800 µL of cold 10% (v/v) glycerol, place tubes back on ice.

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8. Add 50 µL of cells to new 0.5 mL tubes, place back on ice. 
 

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9. Add 1 µL of the plasmid of interest (200 ng/uL to 300 ng/uL; directly from a miniprep should work) to its appropriate tube of cells, place back on ice and mix gently by pipetting .
 

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10. Transfer the cells to cold 2 mm electroporation cuvettes, place back on ice. 
 

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11. Prepare the recovery media in advance by placing 150 µL of Room temperature SOC medium to one 15 mL culture tube for each electroporation.

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12. Turn on the electroporator and create an electroporation program with the following settings: 

Voltage= 2400V
Capacitance = 25 μF 
Resistance = 200 Ω 
Cuvette size = 2 mm
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13. Fully dry the cuvette before placing it into the electroporator chamber. 
 

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14. Electroporate your cuvette.
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15. As quickly but as gently as possible, remove a little less than 150 µL of SOC medium from the appropriate tube for the plasmid and pipette it into the cuvette. Remove the full volume from the cuvette and return it to the original culture tube. 
 


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16. Incubate culture tubes at 28 °C, shaking at 225 rpm, 240 min. 
 


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17. Add 4-6 sterile glass beads to each LB plate.
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18. Add 10 µL cells to the appropriate plates. 
 


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19. Seal and invert the plates and incubate them at 28 °C for about 4 days.