Jan 24, 2025
Bi-parental conjugation protocol for non-model bacteria using E.coli WM3064 DAP auxotroph donor
- 1The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, Tromsø, Norway;
- 2Microalgae & Microbiomes Research Group (M2RG), UiT - The Arctic University of Norway, Tromsø, Norway;
- 3The Arctic Centre for Sustainable Energy (ARC), UiT - The Arctic University of Norway, Tromsø, Norway
Protocol Citation: Atharva Karde, Hans Christopher Bernstein 2025. Bi-parental conjugation protocol for non-model bacteria using E.coli WM3064 DAP auxotroph donor. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3qbzqv25/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 have used this protocol successfully with Arctic marine diatom-associated bacterial recipients.
Created: January 16, 2025
Last Modified: January 24, 2025
Protocol Integer ID: 118568
Keywords: conjugation, bi-parental mating, bi-parental, plasmid, transfer, DAP auxotroph, non-model bacteria
Funders Acknowledgements:
Kometen – Nord-Norges forskningsfond
Abstract
This is a protocol for using bi-parental conjugation to transfer a plasmid into non-model recipient bacteria from a DAP auxotroph donor strain.
Guidelines
- Recipients : donor ratio. For fast-growing commercial strains, the recipient : donor ratio can be 1 : 1, but non-model/slow growing strains will be rapidly out-competed by the donor. In such cases, a larger recipient : donor ratio will give significantly better results. 10 : 1 or even 100 : 1 are some variations of mixtures worth trying.
- Conjugation time. This may need to be optimized based on the recipient strain used. Some may require 24+ hours of incubation. Tweak the mixture ratio and conjugation time such that the donor strain does not out-compete the recipient cells entirely. The incubation temperature can also be regulated to control this.
Materials
1. Donor strain carrying the desired plasmid to be transferred. Your plasmid must contain an oriT sequence in order to be transferred. The donor strain (if using a different one than WM3064) must carry rp4-based transfer machinery and must be 2,6-Diaminopimelic acidThermo Scientific auxotrophic.
2. Recipient strain
3. Growth media for individual strains. Donor growth media must contain 0.3mM DAP.
4. Non-selective plates containing DAP (0.3 mM) for conjugation. These plates should be made from media that both strains can grow on, and contain no antibiotic.
5. Selective plates containing the same antibiotic as the marker on the plasmid you want to transfer, but no DAP. To plate dilutions, you need 3-4 plates per conjugation mixture.
6. 1X PBS (Phosphate-buffered saline )
7. 2,6-Diaminopimelic acidThermo Scientific
8. Pipettes, microcentrifuge tubes, and incubator that supports growth of both donor and recipients.
9. Antibiotics for plasmid selection
| A | B | |
| Antibiotic | Working Concentration | |
| Ampicillin | 100 µg/ml | |
| Carbenicillin | 100 µg/ml | |
| Chloramphenicol | 33 µg/ml | |
| Kanamycin | 50 µg/ml | |
| Streptomycin | 25 µg/ml | |
| Tetracycline | 15 µg/ml |
Protocol materials
1X PBS (Phosphate-buffered saline )
1X PBS (Phosphate-buffered saline )
1X PBS (Phosphate-buffered saline )
1X PBS (Phosphate-buffered saline )
1X PBS (Phosphate-buffered saline )
2,6-Diaminopimelic acidThermo Scientific
1X PBS (Phosphate-buffered saline )
2,6-Diaminopimelic acidThermo Scientific
2,6-Diaminopimelic acidThermo Scientific
2,6-Diaminopimelic acidThermo Scientific
2,6-Diaminopimelic acidThermo Scientific
Before start
1. Verify that the donor strain is carrying the desired plasmid to be transferred
2. Your plasmid MUST CONTAIN an oriT sequence in order to be transferred.
3. The donor strain (if using a different one than WM3064) must carry rp4-based transfer machinery and must be 2,6-Diaminopimelic acidThermo Scientific auxotrophic in order to be eliminated during selection.
4. Ensure that appropriate growth medium is used to grow individual strains and that the donor strain growth medium contains 0.3 millimolar (mM) 2,6-Diaminopimelic acidThermo Scientific .
5. Non-selective plates must contain DAP. These plates should be made from media that both strains can grow on, and contain no antibiotic. You can set up ~4 conjugation mixtures per plate.
6. Selective plates must contain NO DAP and the appropriate antibiotic depending on the selection marker used in the transferred payload. You may also include additional counter selectable markers to minimize E. coli background.
This protocol was developed as part of a master's thesis affiliated with the Microalgae & Microbiomes Research Group at NFH, UiT, Norway.
M2RG Website : https://uit.no/research/micro
Overnight culture preparation
Overnight culture preparation
Inoculate a loopful of donor E.coli WM3064 and recipient strains in respective culture tubes containing 5 mL of growth medium. Ensure that the donor strain is grown in presence of 0.3 millimolar (mM) 2,6-Diaminopimelic acidThermo Scientific and appropriate antibiotic according to the selection marker present on the carried plasmid.
Incubate the cells Overnight or until they have grown sufficiently. The recipient cells may take longer to grow.
Conjugation mixture preparation
Conjugation mixture preparation
5m
5m
Transfer 1 mL of donor and recipient overnight cultures to microcentrifuge tubes.
Gently spin down the cells at 3000 rpm, 00:05:00 and wash with 500 µL 1X PBS (Phosphate-buffered saline ) .
5m
Repeat go to step #4 .
Resuspend the cells in 500 µL 1X PBS (Phosphate-buffered saline ) .
Note
Washing removes residual antibiotic from the donor cell culture. Recipient cells can also be scraped off of plates and washed in PBS.
Prepare 100 µL conjugation mixture by combining the desired volume ratio of donor and recipient cells in a microcentrifuge tube. Mix by pipetting twice.
Note
For fast-growing commercial strains, the recipient : donor ratio can be 1 : 1, but non-model/slow growing strains will be rapidly out-competed by the donor. In such cases, a larger recipient : donor ratio will give significantly better results. 10 : 1 or even 100 : 1 are some variations of mixtures worth trying.
Non-selective plating
Non-selective plating
Spot-plate 100 µL of the mixture onto a non-selective plate containing DAP and no antibiotic. DO NOT SPREAD.
Note
Up to 4-5 conjugation mixtures can be set up on the same plate by carefully plating into quadrants on the plate.
Let the spots dry and incubate the plates Overnight .
Note
This step may need to be optimized based on the recipient strain used. Some may require 24+ hours of incubation. Tweak the mixture ratio and conjugation time such that the donor strain does not out-compete the recipient cells entirely. The incubation temperature can also be regulated to control this.
Washing
Washing
5m
5m
Scrape each conjugation mixture from the non-selective plates and resuspend in 1 mL 1X PBS (Phosphate-buffered saline )
Vortex and gently spin down 3000 rpm, 00:05:00 .
5m
Resuspend in 1 mL 1X PBS (Phosphate-buffered saline )
Repeat go to step #11
Resuspend the mixture in 100 µL 1X PBS (Phosphate-buffered saline )
Selective plating
Selective plating
Spread-plate the entire 100 µL or desired dilutions of washed mixtures on selective plates containing antibiotic and no DAP.
Incubate the plates according to the recipient strain requirements and pick single colonies to restreak or grow in selective media.
Confirm the transfer of the payload via PCR.
Protocol references
Acknowledgements
This protocol was developed as part of a master's thesis affiliated with the Microalgae & Microbiomes Research Group at NFH, UiT, Norway.
M2RG Website : https://uit.no/research/micro
The donor strain used in this project was generously provided by the Lowe-Power Lab at UC Davis (https://lowepowerlab.ucdavis.edu/).