Jul 01, 2025
Unmarked_Gene_Deletion_Ralstonia_Electroporation_Transformation
- Noah Guillome1
- 1Lowe-Power Lab, UC Davis
Protocol Citation: Noah Guillome 2025. Unmarked_Gene_Deletion_Ralstonia_Electroporation_Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8q3z6l2w/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: June 25, 2025
Last Modified: July 01, 2025
Protocol Integer ID: 221076
Keywords: Cloning, Unmarked Deletion, Ralstonia, pUFR80, Targeted Gene Knockout, Gene Deletion, Homologous Recombination, Genome Editing, Chromosomal Integration, Electroporation, Kanamycin Resistance, SacB counter-selection, pufr80 plasmid into ralstonia, unmarked gene deletion in ralstonia, modified pufr80 plasmid, pufr80 plasmid, deletion mutant, desired deletion mutant, unmarked gene deletion, plasmid integration via homologous recombination, selection on kanamycin, homologous recombination, plasmid, gene, mutant, following plasmid integration, recovery of kanamycin, second recombination event, ralstonia, preparation for downstream pcr, kanamycin, electroporation
Abstract
This protocol describes the electroporation-based transformation of a modified pUFR80 plasmid into Ralstonia, as part of the workflow for an unmarked gene deletion in Ralstonia. It includes selection on kanamycin, counter-selection on sucrose, and preparation for downstream PCR and sequencing validation.
The expected outcome is recovery of kanamycin-resistant colonies following plasmid integration via homologous recombination. A second recombination event enables sucrose resistance, yielding either wild-type revertants or the desired deletion mutants.
Guidelines
This protocol is intended for generating an unmarked gene deletion in Ralstonia using the pUFR80 vector backbone. It assumes the pUFR80 with deletion construct was assembled using the Unmarked_Gene_Deletion_Ralstonia_Gibson_Assembly_&_Transformation protocol, subsequently validated by the Unmarked_Gene_Deletion_Ralstonia_Colony_PCR protocol. If modifications were made from either of those protocols, transformation efficiency into Ralstonia may be reduced or fail altogether.
Materials
Reagents
- Sequence-confirmed pUFR80 plasmid (e.g., pUFR80 with deletion construct)
- Electrocompetent Ralstonia cells (same strain background used in gene deletion)
- CPG liquid medium (per liter: 1 g Casamino acids, 10 g Bacto-Peptone, 5 g Glucose (Dextrose), 1 g Yeast extract, and fill to 1000 ml total with Milli-Q water)
- CPG agar (500 µl CPG liquid medium with 7.5 g agarose)
- TZC (Triphenyltetrazolium chloride)
- Kanamycin 25 mg/ml
- Sucrose (7.5% final concentration)
- Autoclaved Milli-Q water
- Nuclease-free water
- 75% glycerol
Equipment
- Nitrile gloves
- PCR tubes
- 1 mm electroporation cuvettes
- Electroporator (settable to 900 V)
- Ice bucket and ice
- 2.0 ml microcentrifuge tubes
- Thermocycler
- Benchtop shaker incubator (28°C, 250 rpm)
- Pipettes (P2, P20, P200, P1000) and sterile tips
- Sterile glass beads or sterile hockey stick (for spreading cells)
- Parafilm
Troubleshooting
Safety warnings
- Troubleshooting Failure (No Growth on Kanamycin): If no colonies appear after kanamycin selection, likely causes include: A. Delayed handling during electroporation (see Transformation Speed below). B. Low-density electrocompetent cells. C. Poor electroporation pulse (e.g., improper voltage or arc discharge). In these cases, repeat the transformation using freshly prepared competent cells and verify plasmid concentration and pulse quality.
- False Positives (Growth on Kanamycin During Final Screen): If colonies grow on kanamycin after sucrose counter-selection, they may still carry the plasmid in a merodiploid state (first recombination only). Only isolates that fail to grow on kanamycin should move forward to colony PCR. If too few pass this screen, sucrose counter-selection may have been ineffective and it is necessary to check media preparation and sucrose concentration (should be 7.5%). If media prep is correct, return to backup kanamycin-resistant stocks, streak on non-selective plates, inoculate liquid culture in non-selective media, and continue with plating onto 7.5% sucrose. Avoid reusing backups more than once or twice and beyond that, restart the transformation process.
- Sucrose Growth Delay: Ralstonia grows slowly on 7.5% sucrose plates and may take a variable amount of time ranging from 2-3 days in the incubator to become visible and ready for the next step. Make sure to plan accordingly around this possibility.
- Transformation Speed: Work quickly once cells thaw to improve transformation efficiency. A. Pre-calculate plasmid volumes based on concentration. B. Label recovery tube in advance. C. Add plasmid immediately after thawing cells. D. Preset pipettes with required volumes. E. Prepare 1000 µl CPG during electroporation to add once electroporator signals the end of the pulse.
Before start
Follow validation steps outlined in Unmarked_Gene_Deletion_Ralstonia_Colony_PCR to confirm modified pUFR80 contains the deletion construct.
Electroporation Transformation
Transformation Setup
Retrieve sequence-confirmed plasmid from the Unmarked_Gene_Deletion_Ralstonia_Colony_PCR protocol, stored at -20ºC. Thaw on ice.
Place a 1mm electroporation cuvette on ice.
Retrieve electrocompetent Ralstonia cells (same strain background used for gene deletion) from -80ºC. Thaw on ice.
Electroporation
Add 1000 ng of sequence-confirmed plasmid (e.g., 10 µl if 100 ng/µl) to the competent cells. Mix by gently flicking the tube.
Transfer 200 µl of the mixture into the chilled electroporation cuvette.
Wipe outside of cuvette with a Kimwipe.
Insert cuvette into the electroporator (dent toward the back).
Set electroporator to 900 volts and pulse.
Recovery
After the pulse (signaled by a beep), add 1000 µl CPG directly to the cuvette.
Resuspend by pipetting up and down twice.
Transfer full volume to a 2 ml microcentrifuge tube.
Incubate at 28ºC shaker incubator (250 rpm) for 4 hours.
Dilution and Plating
Take the recovered Ralstonia cells from the outgrowth and prepare a 10 fold dilution and 100 fold dilution of the cells.
Label 4 CPG + 1% TZC + kanamycin plates as follows:
- Plate 1: 100 µl undiluted
- Plate 2: 100 µl of 10-1 dilution
- Plate 3: 100 µl of 10-2 dilution
- Plate 4: Pellet remaining cells, discard supernatant, resuspend pellet, and plate residual volume
Plate Handling
Spread evenly with sterile beads or hockey stick.
Allow plates to dry.
Incubate for 3-4 days at 28ºC.
Liquid Culture Preparation
A. Prepare a test tube with:
- 3 mL CPG broth
B. Inoculate with an isolated colony, choosing from the colonies with kanamycin resistance
C. Incubate overnight in shaker incubator at 28ºC and 250 rpm.
SacB Counter-selection
Backup Kanamycin Resistant Stock
Use 500 µl of the overnight culture prepared the day prior to make backup glycerol stocks (500 µl culture + 500 µl 75% glycerol).
Store at -80ºC
Dilution and Plating
Prepare a 10 fold, 100 fold, and 1000 fold dilutions of the liquid culture, using autoclaved Milli-Q water to dilute.
Label 3 CPG + 1% TZC + 7.5% sucrose plates as follows:
- Plate 1: 100 µl of 10-1 dilution
- Plate 2: 100 µl of 10-2 dilution
- Plate 3: 100 µl of 10-3 dilution
Plate Handling
Spread evenly with sterile beads or hockey stick.
Allow plates to dry.
Incubate for 2-3 days at 28°C.
Loss of Kanamycin Resistance Screen
Patch Plating
Select 24 diverse colonies from sucrose plates.
For each isolate:
- Streak a lawn onto CPG + 1 %TZC (4 isolates per plate).
- Using the same stick, patch streak onto CPG + 1% TZC + kanamycin plate (24 isolates total).
- Use new stick per isolate to streak from lawn and fill out a quadrant of the CPG + 1% TZC plate.
- Incubate for 2 days at 28ºC.
Liquid Culture Preparation
For isolates that do not grow on kanamycin:
A. Prepare a test tube with:
- 3 mL CPG broth
B. Inoculate with an isolated colony from the CPG + 1% TZC plate.
C. Incubate overnight at 28°C, shaking at 250 rpm.
These will be used for making backup glycerol stocks as well as colony PCR for a final external PCR screen.