Preparation of electrocompetent cells

Writing/editing credits: Tiffany Lowe-Power

Following this protocol should yield enough cells for ~20 electro-transformations. If properly prepared and stored in -80, cells should be viable for over 6 months.

1. Inoculate a single colony from a fresh plate (2-3 days of incubation at 28C) into 5 ml of CPG in a 14 mm test tube and incubate overnight at 28°C and 250 rpm.

2. Add 50 ul of the overnight culture to 100 ml CPG in 250 ml flask. Note: If performed around 5pm, the culture will be mid-log (OD 0.5 to 0.7) by 9 to 10am the following day. Make sure you have 10% glycerol in the 4 °C fridge for the next morning. *_Second note:_ For GMI1000, PSI07, and IBSBF1503, diluting your overnight cultures to an OD600 of 0.02, adding 1 mL of this suspension into 100 mL of CPG broth, and incubating (28c, 250 rpm) this culture around 5pm results in an OD of about 0.3 (the range was .29-.34) at 9am the next morning

3. Shake at 28°C and 250 rpm until the culture reach mid-log (OD600 of 0.5-0.7).

4. Place culture on ice for 10 min. From this point keep the cultures ice cold. Pour the culture into chilled 50 ml conical tubes.

5. Centrifuge at 5000 xg (xxx rpm in xxx rotor) for 10 min. Carefully pour off the supernatant and aspirate any residual broth. For EPS⁺ Ralstonia cells, it may not be possible to aspirate the residual broth without loss of cells. If so, make sure to perform an extra 10% glycerol wash.

6. Add 10 ml of cold 10% glycerol to cell pellet & resuspend the cells by pipetting up and down. To pool the pellets between tubes, use this glycerol cell suspension to resuspend the second pellet. Transfer to 15 ml tube.

7. Centrifuge at 5000 xg for 10 min.

8. Pour off the supernatant. Resuspend the cells in 6 ml glycerol and re-centrifuge.

9. Remove supernatant by pipetting (EPS+ Ralstonia strains will have a loose pellet) and suspend the cells in the residual glycerol by pipetting up and down.

10. Cells can aliquoted and saved in -80 or used immediately for electroporation. To freeze, add 80 ul of the culture to 1.5 ml tubes on wet ice. Once aliquoted, transfer the tubes to dry ice for ~10 minutes before transfer them to a -80°C freezer. The cultures should be good for >6 months. Date the outside of the box or include a paper tag with the cells to indicate their “Best By” date.

Electroporation Protocol

1. Turn on the electroporator and set to 900 V.
   • Note: We don’t adjust ohms/µF on our electroporator, but these conditions worked in Lindow lab: 0.9 kv, 400 ohms and 25 µF.

2. Thaw cells on ice for 10 min or use freshly made cells on ice.

3. Add up to 8 µL of plasmid DNA (approximately 2 µg) to cells.
   • Note: This volume of plasmid has not been optimized, but we have seen more success using a larger amount of plasmid up to 20uL of volume

4. Gently pipette to mix and transfer the DNA-cell mixture to a 1 mm electroporation cuvette, tap on countertop 2x to reduce bubbles, dry exterior of cuvette, and place in the electroporator & shock.

5. ~Immediately add 1 ml of CPG broth (no antibiotics) to electroporation cuvette, mix gently by slowly pipetting up and down once and transfer mixture to a pre-labeled 2.0 ml tube.

6. Immediately incubate with shaking at 28C for ~4 hr to allow cells to recover (Lie tube ~horizontally e.g. in an empty Erlenmeyer flask). Pre-warm CPG +TZC +antibiotic plates at 28°C.

7. Dilution plate onto CPG-antibiotic media. Plate 200 ul of undiluted, 1:10, 1:100 and 1:1000 diluted cells. Use sterile glass beads to spread. Then dump the beads in a “dirty beads” reservoir.
   • To reuse beads: Add 10% bleach to the dirty beads container. Incubate 30 min. Then dump beads into a metal strainer. Rince very well with water. Line a plastic bin with a paper towel. Dump in wet beads. Allow to dry overnight. Use a funnel to pour beads into small glass bottles. Label with autoclave tape & autoclave with a dry cycle. 8433
   • Optional: spin down the remaining culture & resuspend in 200 ul to spread on a plate.
   • Optional control: plate some of the bacteria on CPG without antibiotics to verify viability & verify the antibiotic plates are appropriately selective.

8. Incubate 2-3 days at 28 C and look for single colonies (occasionally colonies take up to 4 days to appear and subsequently grow normally after the first selection).

9. Restreak 1-3 single colonies and incubate ~2 days. Check genotype with [colony PCR].
   • Colony PCR note: Colony PCR is more successful after overnight incubation of colonies in CPG broth than picking colonies straight into a PCR reaction… maybe because of relative amounts of EPS?

Re-use:

• Reuse the 4.5 mm glass beads. After spread plating, dump beads in a beaker and sterilize with 10% bleach. Rinse with diH2O and autoclave for re-use.

• Consider washing and re-using the electroporation cuvettes only if you find a suitable protocol that seems to clean them without compromising results.