Mar 25, 2026
Synechococcus culture and transformation
- Yibo Wu1
- 1UNSW
Protocol Citation: Yibo Wu 2026. Synechococcus culture and transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jzjkvqe/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: March 25, 2026
Last Modified: March 25, 2026
Protocol Integer ID: 313872
Keywords: transformation protocol for synechococcus culture, synechococcus culture, transformation protocol
Abstract
Protocol for Synechococcus culture and transformation
Guidelines
**General guidelines for Synechococcus elongatus PCC 7942 culture**
- Use proper aseptic technique and work in a laminar flow hood.
- Grow cells using Gibco™ BG-11 medium (Cat. nos. A1379901, A1379902).
- Grow S. elongatus liquid cultures at 34°C ± 1°C with CO₂ (1–2% in air) under continuous illumination using moderate light intensities.
- S. elongatus cultures can also be grown using light intensities of 50–400 μE m⁻² s⁻¹ with only atmospheric CO₂.
- The presence of CO₂ is needed for optimal growth.
- S. elongatus solid cultures on BG-11 agar plates can be grown at 34°C under continuous illumination using 100–200 μE m⁻² s⁻¹ of cool fluorescent white light.
- Optimal equipment for culturing S. elongatus is an algal growth chamber.
- Do not stack culture plates to allow continuous uniform illumination.
- Grow cells until the culture reaches OD₇₅₀ of ≥1 before transformation.
- Take OD measurements at 750 nm.
- S. elongatus is classified as a GRAS organism. Follow general safety guidelines under Biosafety Level 1 (BL-1) containment.
**Guidelines for thawing**
- Do not thaw more than 3 vials at a time.
- Frozen S. elongatus cells are very sensitive to temperature fluctuations.
- Before the cells are thawed, the cells must be transferred from the −80°C freezer into a dry ice container as quickly as possible and the vials should be buried in dry ice.
- To maximize the recovery of the cells when thawing, warm the cells very quickly by placing the tubes directly from the dry ice container into a 34°C water bath. When the cells are completely thawed, immediately dilute them into Gibco™ BG-11 medium, pre-warmed to room temperature.
- You can count the S. elongatus cells using the Attune™ NxT Acoustic Focusing Cytometer (or equivalent) by detecting the endogenous orange-fluorescent phycoerythrin and red-fluorescent chlorophyll. For more information, contact Cell Analysis Technical Support Center (www.thermofisher.com/support).
**Guidelines for transformation**
- Synechococcus elongatus is naturally transformable with highest efficiencies of transformation when the culture is in the log phase of growth (OD₇₅₀ of 1–2).
- Transform S. elongatus using circular, supercoiled DNA.
- Incubate the transformation reaction at 34°C, in the dark.
Note: Darkness increases the transformation efficiency.
- The quality and the concentration of the plasmid DNA play a central role for the efficiency of transformation. Use a commercial kit such as the PureLink™ HQ Mini Plasmid Purification or the PureLink™ HiPure Plasmid Miniprep kits that deliver pure DNA (see page 19 for ordering information).
- Pre-warm the selective BG-11 + spectinomycin plates to room temperature for 1 hour before plating the transformants.
Materials
**LB (Luria-Bertani) medium and plates**
**LB medium**
1. 10 g tryptone
2. 5 g yeast extract
3. 10 g NaCl
4. NaOH
5. Spectinomycin
**LB agar plates**
1. LB medium
2. 15 g/L agar
**BG-11 agar plates**
1. 15 g agar
2. Gibco™ BG-11 medium (Cat. No.12177, Sigma, Cat. No. A1296)
3. Spectinomycin
**Materials needed**
- 34°C water bath with a dark lid or covered with aluminum foil
- Algal growth chamber (e.g., Percival™ Algal Chamber from Geneva Scientific) set to 34°C ± 1°C and 1% CO₂, optimal, under continuous illumination with 50 μE m⁻² s⁻¹
Note: If an algal growth chamber is not available, you can use a standard cell culture incubator under continuous illumination using moderate light intensities of cool fluorescent white (50 μE m⁻² s⁻¹).
- Gyratory shaking platform set to 100 rpm
- 6-well clear-bottom culture plates
- Gibco™ BG-11 medium (Cat. No. A1379901 or A1379902), pre-warmed to room temperature
- 70% ethanol
- Dry ice
- pSyn_6 construct carrying your gene of interest (purified, supercoiled plasmid DNA from page 9)
- Optional: pSyn_6 positive control construct carrying the S. elongatus codon-optimized GUS gene
Note: You can order synthesized codon-optimized genes from Invitrogen™ GeneArt™ Gene Synthesis services (www.thermofisher.com/geneartsynthesis).
- BG-11 agar plates containing 10 μg/mL spectinomycin, pre-warmed to room temperature (see page 10 for recipe)
Note: You will need 2 plates per transformation.
- Sterile microcentrifuge tubes
- Disposable spreaders
Troubleshooting
Safety warnings
IMPORTANT! Do not incubate under light intensity of more than 50 μE m⁻² s⁻¹ because the cells are sensitive to light immediately after resuscitation. Do not stack the plates.**
Appendix A: Support protocols
LB medium
For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL of deionized water.
Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to 1 liter.
Autoclave the solution on liquid cycle for 20 minutes at 15 psi. Allow the solution to cool to 55°C and add the appropriate antibiotics, if needed. Note: Use spectinomycin at a final concentration of 100 µg/mL.
Store the medium at room temperature or at 2–8°C.
LB agar plates
Prepare LB medium as above, but add 15 g/L agar before autoclaving.
Autoclave the medium plus agar on liquid cycle for 20 minutes at 15 psi.
After autoclaving, cool the medium to ~55°C, add the appropriate antibiotics, and pour into 10-cm plates.
Let the agar harden, then invert the plates and store them at 2–8°C, in the dark.
BG-11 agar plates
Add 15 g of agar to 200 mL of Gibco™ BG-11 medium in an autoclavable flask. Note: Use high quality agar, such as Calbiochem, Cat. No.12177, or Sigma, Cat. No. A1296.
Autoclave on liquid cycle for 20 minutes.
Warm 800 mL of Gibco™ BG-11 medium to 55–60°C in a water bath.
After autoclaving, cool the agar containing flask to ~55°C.
Combine the agar containing flask with 800 mL of Gibco™ BG-11 medium.
Add spectinomycin to a final concentration of 10 µg/mL (if required), and pour into 10-cm plates.
Let the plates harden (do not over dry), invert them, and store at 2–8°C in the dark. Final agar concentration will be 1.5%.
Guidelines for Synechococcus elongatus PCC 7942 culture
The following are culture guidelines for the cyanobacterium Synechococcus elongatus PCC 7942. All solutions and equipment that may contact cells must be sterile. Always use proper aseptic technique and work in a laminar flow hood.
Grow the cells using Gibco™ BG-11 medium (Cat. nos. A1379901, A1379902), which is specifically formulated for optimal growth and maintenance of S. elongatus cells.
Grow S. elongatus liquid cultures at 34°C ± 1°C with CO₂ (1–2% in air) under continuous illumination using moderate light intensities of cool fluorescent white (50–100 µE m⁻² s⁻¹) with agitation on a gyratory shaker set to 100 rpm.
Note: S. elongatus cultures can also be grown using light intensities of 50–400 µE m⁻² s⁻¹ with only atmospheric CO₂ (i.e., without additional CO₂).
The presence of CO₂ is needed to obtain optimal growth of S. elongatus in liquid culture as it is a photosynthetic organism; however, additional CO₂ is not necessary during transformation where the cells are grown on agar support and are exposed to atmospheric CO₂.
If you are bubbling the culture with CO₂ enriched air or CO₂ gas, prepare the BG-11 medium with 50 mM NaHCO₃ and adjust the pH to 7.5. The presence of NaHCO₃ in the medium prevents it from becoming acidic.
S. elongatus solid cultures on BG-11 agar plates can be grown at 34°C under continuous illumination using 100–200 µE m⁻² s⁻¹ of cool fluorescent white light.
Note: You can also incubate the cultures at room temperature if sufficient light is provided; however, the growth will be slower.
The optimal equipment for culturing S. elongatus is an algal growth chamber (e.g., Percival™ Algal Chamber from Geneva Scientific) with regulatable light supply and a light meter to guide adjustments.
If an algal growth chamber is not available, the cells can be grown in a standard cell culture incubator illuminated with cool fluorescent lights placed within 12 inches of the culture plates. Standard room lights and incubation at room temperature provide sub-optimal growth conditions.
Do not stack the culture plates to allow continuous uniform illumination.
Grow the cells until the culture reaches OD₇₅₀ of ≥1 before transformation.
Take the OD measurements at 750 nm.
S. elongatus is classified as a GRAS (generally regarded as safe) organism with no known viral or bacterial pathogens.
Thaw Synechococcus elongatus PCC 7942
Do not thaw more than 3 vials at a time.
Frozen S. elongatus cells are very sensitive to temperature fluctuations.
Before the cells are thawed, the cells must be transferred from the −80°C freezer into a dry ice container as quickly as possible and the vials should be buried in dry ice.
To maximize the recovery of the cells when thawing, warm the cells very quickly by placing the tubes directly from the dry ice container into a 34°C water bath. When the cells are completely thawed, immediately dilute them into Gibco™ BG-11 medium, pre-warmed to room temperature.
You can count the S. elongatus cells using the Attune™ NxT Acoustic Focusing Cytometer (or equivalent) by detecting the endogenous orange-fluorescent phycoerythrin and red-fluorescent chlorophyll. For more information, contact Cell Analysis Technical Support Center (www.thermofisher.com/support).
Thaw procedure
Remove the frozen cells from −80°C storage and immediately place them in a dry ice container. Bury the vial(s) containing the cells in dry ice to minimize temperature fluctuations before thawing.
Add 6 mL of Gibco™ BG-11 medium, pre-warmed to room temperature, into each well of a 6-well plate.
Remove the cryovial containing the frozen cells from the dry ice storage and immediately place it into a 34°C water bath.
Quickly thaw the cells by placing the vial containing the cells in the 34°C water bath until the last ice crystal has melted (~2 minutes). Do not agitate the cells while thawing (i.e., do not swirl the vial).
After the cells have thawed, wipe the outside of the vial with 70% ethanol, and place the vial in a rack at room temperature. Proceed immediately to the next step.
Transfer 100 µL of thawed cells from the vial into each well of the 6-well plate containing 6 mL of Gibco™ BG-11 medium.
Place the 6-well plates in the algal growth chamber set to 34°C ± 1°C with 1% CO₂ and illuminated with constant light of 50 µE m⁻² s⁻¹.
IMPORTANT! Do not incubate under light intensity of more than 50 µE m⁻² s⁻¹ because the cells are sensitive to light immediately after resuscitation. Do not stack the plates.
Incubate the cells with gentle agitation on a gyratory shaker set to 100 rpm.
On Day 2, transfer 400 µL of the cell suspension and into a disposable plastic cuvette containing 400 µL of Gibco™ BG-11 medium to measure the optical density.
Measure the cell density at 750 nm (OD₇₅₀). If the OD₇₅₀ is greater than 1, proceed to the transformation step (page 14). If the culture has not yet reached OD₇₅₀ = 1, return it to the algal growth chamber and continue the incubation.
Transform Synechococcus elongatus PCC 7942
Synechococcus elongatus is naturally transformable with highest efficiencies of transformation when the culture is in the log phase of growth (OD₇₅₀ of 1–2).
Transform S. elongatus using circular, supercoiled DNA.
Incubate the transformation reaction at 34°C, in the dark.
Note: Darkness increases the transformation efficiency.
The quality and the concentration of the plasmid DNA play a central role for the efficiency of transformation. Use a commercial kit such as the PureLink™ HQ Mini Plasmid Purification or the PureLink™ HiPure Plasmid Miniprep kits that deliver pure DNA (see page 19 for ordering information).
Pre-warm the selective BG-11 + spectinomycin plates to room temperature for 1 hour before plating the transformants.
Measure the optical density of the Synechococcus elongatus cultures at 750 nm (i.e., OD₇₅₀). Note: For best performance, the OD₇₅₀ of cultures should be greater than 1 and less than 2.
Harvest 1.5 mL of the cells (per transformation) by centrifugation at 14,000 rpm for 3 minutes at room temperature.
Remove the supernatant by pipetting.
Resuspend the cells in 1 mL of Gibco™ BG-11 medium by gently pipetting up and down.
Centrifuge the cells at 14,000 rpm for 1 minute at room temperature, and remove the supernatant by pipetting.
Resuspend the cells in 100 µL of Gibco™ BG-11 medium by gently pipetting up and down.
Add 100 ng of supercoiled plasmid DNA (i.e., pSyn_6 construct containing your gene of interest) into the resuspended cells. In a separate tube, add 100 ng of empty pSyn_6 vector as a negative control. Mix the DNA-cell suspension gently by flicking the tube.
Incubate the cell-DNA mixtures in the 34°C water bath with a dark lid for 4 hours. After the incubation is complete, remove the tubes from the water bath and wipe them with 70% ethanol.
Plate 80 µL and 5 µL of each transformation mixture on separate BG-11 agar plates containing 10 µg/mL of spectinomycin and pre-warmed to room temperature.
Place the plates with agar side down on illuminated shelves at room temperature (25–30°C). Do not stack the plates to ensure continuous and even illumination.
Incubate the plates for 5–7 days or until the colonies are ready to pick. The results from the transformation with the pSyn_6 construct will depend on the nature of your gene of interest.
Protocol references
For more information on BL-1 guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th ed._, published by the Centers for Disease Control, which is available for download from www.cdc.gov.