Jul 07, 2025

Public workspaceElectroporation - mediated transformation protocol for marine isolated bacteria

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzqwmrvx1/v1
Electroporation - mediated transformation protocol for marine isolated bacteria
  • William Arnli1,2,
  • Johan Bjerg1,2,
  • Hans Christopher Bernstein1,2,3
  • 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
William Arnli
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzqwmrvx1/v1
Protocol Citation: William Arnli, Johan Bjerg, Hans Christopher Bernstein 2025. Electroporation - mediated transformation protocol for marine isolated bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzqwmrvx1/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 20, 2025
Last Modified: July 07, 2025
Protocol Integer ID: 220626
Keywords: Electroporation , Transformation , Marine Isolated Bacteria , Plasmid DNA, Biotechnology , Electrocompetent cells, Transformation protocol, Genetic transformation, Marine bacteria, Marine microbiology, Synthetic biology, Plasmids, Competent cells, Gene transfer, Genetic engineering, Plasmid DNA, Transformation efficiency, Competent cell preparation, Cell viability, Osmotic stress, Transformation protocol optimization, Antibiotic selection, Kanamycin selection, Sulfitobacter marinus, Fast-growing strains, Slow-growing strains, Broad-host-range plasmids, bacteria electroporation protocol, isolated bacteria electroporation protocol, plasmid dna, transforming marine, electroporation, current genetic tool, isolated bacteria, plasmid, limitations of current genetic tool, dna, preparing competent cell, marine application, mediated transformation protocol
Abstract
Electroporation protocol specialized for transforming marine isolated bacteria strains with plasmid DNA, developed to resolve limitations of current genetic tools for marine application. The steps involve preparing competent cells for a single cycle of electroporation.

Guidelines
Optimization of electroporation
This electroporation protocol may need to be optimized depending on variability of strains.


Table 1 - Suggested control plate scheme
ABCDEFG
ID Culture type Plate type Control type uL to add ID number Comments
NC1 Strain_1 no plasmid Antibiotic selection Negative control 200 N1 Should have no growth
NC2 Strain_2 no plasmid Antibiotic selection Negative control 200 N2 Should have no growth
NC3 Strain_3 no plasmid Antibiotic selection Negative control 200 N3 Should have no growth
SC1 Strain_1 + plasmid Growth media Survival control 50 S1 Should have growth. Controls for survival of electroporation procedure
SC2 Strain_2 + plasmid Growth media Survival control 50 S2 Should have growth. Controls for survival of electroporation procedure
SC3 Strain_3 + plasmid Growth media Survival control 50 S3 Should have growth. Controls for survival of electroporation procedure
T1 Strain_1 + plasmid Antibiotic selection Selection control 50 AS1 Only transformed cells should grow
T2 Strain_2 + plasmid Antibiotic selection Selection control 50 AS2 Only transformed cells should grow
T3 Strain_3 + plasmid Antibiotic selection Selection control 50 AS3 Only transformed cells should grow


Materials

MATERIALS and REAGENTS
ReagentBD Difco™ Dehydrated Culture Media: Marine Broth 2216Thermo Fisher Scientific
ReagentPeptone from casein (Tryptone)Merck MilliporeSigma (Sigma-Aldrich)
ReagentAgar Merck MilliporeSigma (Sigma-Aldrich)Catalog #05040
ReagentKanamycin sulphateVWR International (Avantor)
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)

Equipment
Electroporation Cuvettes
NAME
VWR
BRAND
732-1135
SKU
1 mm Gap, 90 μl Gray Sterile
SPECIFICATIONS

Equipment
Heraeus Multifuge 1 S-R
NAME
Sentrifuge
TYPE
Thermo Scientific
BRAND
NA
SKU

Equipment
ECM® 630 Electroporation System
NAME
Electroporation System
TYPE
BTX Harvard Apparatus
BRAND
NA
SKU


MEADIA

FMAP (1 L)
Amount15 g Marine Broth
Amount5 g Peptone
Amount300 mL Filtered seawater
Amount700 mL MilliQ water

FMAP agar 
Amount1 L FMAP medium
Amount15 g Agar

ANTIBIOTIC SELECTION
Recommended stock and working concentrations for antibiotic selection

AntibioticRecommended Stock ConcentrationRecommended Working Concentration
Ampicillin100 mg/mL100 µg/mL
Bleocin5 mg/mL5 µg/mL
Carbenicillin*100 mg/mL100 µg/mL
Chloramphenicol25 mg/mL(dissolve in EtOH)25 µg/mL
Coumermycin25 mg/mL(dissolve in DMSO)25 µg/mL
Gentamycin10 mg/mL10 µg/mL
Kanamycin100 mg/mL100 µg/mL
Spectinomycin50 mg/mL50 µg/mL
Tetracycline10 mg/mL10 µg/mL
Citation
addgene (2016). Pouring LB Agar Plates. https://www.addgene.org/protocols/pouring-lb-agar-plates/.
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LINK

Troubleshooting
Safety warnings
Refer to the Safety Data Sheets (SDS) for information on health and environmental hazards. Be sure to follow your institution's Health and Safety (HMS) guidelines, wear appropriate personal protective equipment (PPE), and adhere to all established safety protocols to minimize risks
Before start
Widely available genetic techniques are primarily designed for model organisms, which exhibit inability to transfer genes or low transformation efficiencies in marine bacteria. This protocol aims to improve transformation efficiency and cell viability, overcoming the challenges with conventional methods in marine application (Zahraa Zeaiter Et al., 2018).

For marine bacteria, high salt concentrations in the growth medium can lead to increased conductivity in the electroporation buffer, which may cause electrical arcing and damage cells, thereby reducing transformation efficiency. However, by simply reducing the salt concentration, issues such as osmotic shock can occur, leading to decrease cell viability (Katrina Christi et al., 2024). Consequently, as osmolytes like glycerol are natural counters for osmotic stress in marine bacteria, Amount10 % glycerol is utilized in this protocol to mitigate this effect.

As many strains and species can have very different growth rates, the protocol differs between fast and slow growers. This definition is vague but the rule of thumb is:

Fast growers reach a stationary-like phase after 12-20 hours in 5 mL of growth medium with shaking at optimal growth temperature (similar to E. coli DH5α e.g.).

Slow growers reach a stationary-like phase after 24-48 hours in growth medium with shaking at optimal growth temperature.

FMAP media was utilized as an all-purpose marine growth media (see Materials for media composition), however it is highly recommended to establish optimal growth media for each bacterial strain. This ensures the highest efficiency in electroporation and recovery of transformants. Additionally, the plasmids utilized in this protocol where based on double kanamycin (Concentration100 µg/ml ) selection, however, selecting the appropriate antibiotics corresponding to the antibiotic selection markers on your plasmids is crucial for proper selection (see Materials for concentration recommendations).

It is also advisable before beginning the procedure, to measure the plasmid concentration (ng/µL) and calculate the necessary volume of plasmid DNA (50-100 ng) required to create competent cells.

Note that preparing and creating competent cells is the most time-consuming step in this protocol. As the protocol provides enough competent cells for a single electroporation cycle, scaling up steps 1 through 9 is recommended to generate enough cells for multiple cycles. Competent cells can be stored at -80°C for future use.

Although transformation has been highly successful for the marine isolated strain Sulfitobacter marinus, with a range of plasmids 1000-4000 base-pairs in size, the electroporation protocol may need to be optimized depending on variability of other marine strains.

This protocol was developed as part of a master's thesis affiliated with Microalgae & Microbiomes - The M2 Research Group at Norges Fiskerihøgskole, University of Tromsø, Norway
Preparation of strains and media
1h 5m
Inoculate strains in Amount7 mL of preferred marine growth media in a Amount15 mL sterile culture tubes and shake DurationOvernight for fast growers or longer for slow growing strains at the strain's optimal growth temperature.
Note
The inoculation time may vary depending on the growth dynamics of your strains, and it is typically considered acceptable for electroporation once the concentration reaches 0.8-1 at OD600.

5m
Incubation
Prepare and transfer Amount10 % glycerol (sterilized), growth medium, growth media plates, growth medium agar plates with appropriate antibiotics, and 1 mm electroporation cuvettes in fridge DurationOvernight



1h
Day 1 - Reviving culture
3h 5m
Using a spectrophotometer, measure the OD600 of the culture.

Note
It is recommended in combination with this step to ensure optimal cell density (0.8 -1 OD600) by measuring OD600.

5m
To revive the overnight cultures, ensuring that the cells transition back into the growth phase, add Amount3 mL growth medium, and return to shaker to let them grow for 2-4 hours


3h
Day 1 - Creating competent cells
58m
Prepare an ice tray and cool a large centrifuge to Temperature4 °C . From this point everything should be kept on ice.

5m
Centrifuge overnight cultures at Centrifigation4500 x g, 4°C, 00:10:00

10m
Discard supernatants and resuspend pellets in Amount5 mL fridge-cooled Amount10 % glycerol

3m
Centrifuge cultures for another Centrifigation4500 x g, 4°C, 00:10:00 and discard supernatants

10m
Go to and repeat until a total 3 washes has been carried out with Amount10 % glycerol. On the final wash, resuspend in Amount100 µL Amount10 % glycerol in sterile microcentrifuge tubes. Store at Temperature-80 °C , or proceed for electroporation.
Note
Preparing and creating competent cells is the most time-consuming step in this electroporation protocol. Since the protocol provides enough competent cells for a single electroporation cycle, it is recommended to scale up steps 1 through 9 to generate enough cells for multiple cycles. The competent cells can be stored at Temperature-80 °C for later use.


30m
Day 1 - Electroporation
2h 40m
Transfer Amount80 µL competent cells (from step 9) to Thikness1 mm electroporation cuvettes.


Note
Remember to include a negative control where plasmid-DNA is not added.
A recommendation for the controls to be included can be found under Guidelines & Warnings.

5m
Add Amount50-100 ng plasmid-DNA to the competent cells. Flick the cuvette 3-4 times to ensure homogeneity, and incubate on ice for 25 minutes.

30m
Insert electroporation cuvettes and electroporate with 1.25 kV, 200 ohms and 25 µF

5m
Critical
Immediately after electroporation add Amount1 mL cold growth medium, homogenize by pipetting up and down.

For fast growers: Transfer to a microcentrifuge tube and incubate for Duration01:30:00 at optimal growth temperature with shaking.

For slow growers: Transfer to a Amount15 mL sterile culture tube, add additional Amount4 mL of growth media and incubate DurationOvernight at optimal growth temperature with shaking.

Note
It might be difficult to extract all the culture from the cuvette. It is recommended to tilt the cuvette to one side allowing the culture to pool to the side while aspirating with a 100 or 200 uL pipette tip.

2h
Critical
Day 1 or 2 - Plating
15m
After recovery, plate the following:

Survival control (SC): Plate Amount50 µL of the transformants on an agar plate with growth medium and no antibiotics. This controls that cells survives the electroporation procedure.

Transformants (T) and negative controls (NC): Plate Amount100 µL of recovery culture on an agar plate with growth medium and appropriate antibiotics.


[Optional]:
If low transformation efficiency is expected, the rest of the culture should be centrifuged atCentrifigation4500 x g, 00:05:00 . Decant supernatant so a little medium remains, re-dissolve cell pellet and plate on appropriate agar plates.

Suggested scheme for plating can be found in Table 1 under Guidelines section.


Note
Alternatively, for calculating transformation efficiency:

Dilute the culture 1000-fold for plating.

Transformation efficiency calculation:

Number of transformants per ug=

(Number of transfomants/ ug of DNA) * (Final volume of recovery (ml) / Volume plated (ml))


15m
Incubate and culture transformants DurationOvernight (or longer if needed) in your preferred marine growth media supplemented with your choice of antibiotics.

Day 2 or 3 - Verification of transformants
Transformants should be verified using a method of choice; for the purpose of this protocol, colony PCR was selected.
Protocol references
Katrina Christi, Jennifer Hudson, Suhelen Egan,
Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency,
Microbiological Research,
Volume 284, 2024,



Zahraa Zeaiter, Francesca Mapelli, Elena Crotti, Sara Borin, Methods for the genetic manipulation of marine bacteria, Electronic Journal of Biotechnology, Volume 33, 2018

Piekarski, T., Buchholz, I., Drepper, T. et al. Genetic tools for the investigation of Roseobacter clade bacteria. BMC Microbiol 9, 265 (2009)

Kyoung-Hee Choi, Ayush Kumar, Herbert P. Schweizer, A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation, Journal of Microbiological Methods, Volume 64, Issue 3, 2006


Citations
addgene. Pouring LB Agar Plates
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