CB2A Electroporation

Objective

Author(s): Jessie Luo, Ada Jiang

While Caulobacter cannot be chemically transformed, it can be electroporated to introduce plasmids into the cells. This protocol describe and electroporation protocol adapted from iGEM British_Columbia 2016, Smit et al., 2000, and a protocol provided by our advisor, Beth.

Materials

Electrocompetent cells
PYE medium
PYE Agar plates (with appropriate antibiotic - e.g. CM 2 µg/mL)
Cuvettes (0.1cm interelectrodal gap)
Electropulser (Eppendorf Eporator®)
Shaking incubator at 30˚C
Plasmid DNA (100-1000 ng/µL)
10 mL aeration/culture tubes (sterile)
Glass beads
Kimwipes / Paper towel
Eppendorf tubes (sterile)

Set-Up

Preparation

Put cuvettes on ice for at least 20 mins to chill (or pre-chill by putting them in the fridge).
Thaw electrocompetent CB2A cells on ice (one tube per plasmid). Do not handle the cells vigorously, as this will reduce transformation efficiency.
(Optional) Thaw DNA samples. Put 1 µL of 100-1000 ng/µL undiluted DNA in new eppendorf tubes (labelled) on ice.
Pre-warm PYE medium by placing a 50 mL aliquot of medium in 30 or 37˚C for about 15 minutes.
Set up a sterile workspace (in the flow hood, BSC or under the flame).
Plug in, turn on and adjust the electropulser to the appropriate settings: 2500 V

Procedure

Electrotransformation

Transfer 50 µL electrocompetent cells to tube with 1 µL DNA on ice. Or directly add the appropriate amount of DNA to the cells.
Wait 1-2 minutes. In this time, dry a cuvette thoroughly with paper, remove its lid and place it in the cuvette holder such that the plastic nose points towards the back.
Transfer the 50 µL DNA/cell mix to the cuvette (careful to place in 1 mm slit) and slide the cuvette holder into the electropulser until you hear a click, confirming the holder is locked in place. Click START to pulse. Uptake 950 µL PYE medium into a p1000 pipette.
Take out the cuvette. Resuspend cells gently, but immediately in 950 µL PYE medium.
Transfer cell suspension to an aeration tube. Use a p200 with gel loading tips to extract remaining cells from the cuvette.
Incubate at 30°C, 200 rpm shaking for 2-4 hours.
Plate on PYE/CM and incubate 30°C for 2-7 days.

Notes on arcing:

When pulsing, the reading gives a number between 1-5 - this is the time constant. 4.5-5 is best.
If arc happens on pulsing (spark etc), then cells are useless - dispose.
Arcing happens if salt concentration is too high. Can dilute the DNA used with water and try again with new cells if this happens.
Ensure cuvettes are ice cold, and wiped properly before pulsing.