May 06, 2026
Transformation of Gardnerella vaginalis ATCC 14018 with vector pG01-MC
- 1Fred Hutch Cancer Center
Protocol Citation: Elliot Lee 2026. Transformation of Gardnerella vaginalis ATCC 14018 with vector pG01-MC. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1o91g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2026
Last Modified: May 06, 2026
Protocol Integer ID: 238673
Keywords: transformation of gardnerella, competent gardnerella vaginalis atcc, atcc 14018 with vector pg01, competent gardnerella, gardnerella, vector pg01, transformation, select for transformant, transformant, containing tetracycline, atcc, mc protocol
Abstract
Protocol to prepare competent Gardnerella vaginalis ATCC 14018, transform it with the vector pG01-MC, and select for transformants using plates containing tetracycline.
Preparation of Competent Cells
3d 16h 50m
Plate 1 µL of G. vaginalis ATCC 14018 frozen stock onto a reduced NYCIII agar plate and incubate anaerobically at 37 °C for 48:00:00 .
2d
Suspend a single colony from the plate into 2 mL reduced NYCIII liquid media and incubate anaerobically at 37 °C for 24:00:00 .
1d
Inoculate 500 µL of the liquid culture into 5 mL reduced NYCIII liquid media and incubate anaerobically at 37 °C for 16:00:00 . This incubation time ensures the bacteria are still actively dividing the next day so it takes less time for them to enter early log-phase growth.
16h
Inoculate 4 mL of the culture into 40 mL reduced Modified de Man-Rogosa-Sharpe Media. Incubate anaerobically at 37 °C until OD600 reaches 0.3-0.4 (usually 4-6 hours). Record the density of the culture at this step so the density of competent cells can be normalized. Note: Transformation efficiency drops approximately 10x if OD600 < 0.3 or OD600 > 0.9.
.
Place culture on ice for 00:20:00 , inverting every 5 minutes. Note: Always keep cells on ice to ensure they remain receptive to plasmid DNA.
20m
Harvest cells by centrifugation at4 °C , 4,052xg for 00:10:00 .
10m
Discard the supernatant, then wash cells by resuspending them in 25 mL ice-cold 10% glycerol solution. Repeat the centrifugation at4 °C , 4,052xg for 00:10:00 .
10m
Repeat the wash by discarding the supernatant, then resuspending the cells in 25 mL ice-cold 10% glycerol solution and centrifuging at4 °C , 4,052xg for 00:10:00 .
10m
Discard the supernatant. Resuspend the cells in maltose-citrate-glycerol buffer (0.105 g citric acid dissolved in 400 mL H2O and 400 mL glycerol, adjust pH to 5.8 using NaOH, adjust volume to 1 L with H2O. Add 85.5 g maltose. Sterilize by autoclaving) to a final OD600 of 40. Aliquot 100 µL of bacterial suspensions in tubes at store at -80 °C .
Transformation
2d 1h
Prewarm reduced NYCIII liquid media to 37 °C for later use.
Aliquot 0.5 µg of HaeIII methyltransferase-treated vector into chilled 1.5 mL tubes. Note: Keep all components on ice until transformation to maximize efficiency.
Thaw frozen competent cells on ice. Once thawed, mixed 50 µL of competent cells with the vector DNA and transfer the entire volume to a pre-chilled, 0.1 cm electroporation cuvette.
Electrotransform the bacteria at 25 μF, 200 Ω, 2000 V then immediately resuspend them in 1 mL prewarmed NYCIII media and recover anaerobically at 37 °C for 01:00:00 .
1h
Plate 100 µL of transformed cells onto NYCIII agar plates containing 16 μg/mL tetracycline. Incubate the plates anaerobically at 37 °C for 48:00:00 to allow transformed cells to grow.
2d