I tried to transform Synechococcus elongatus PCC 7942 by using this protocol.

1. Measure the optical density of the Synechococcus elongatus cultures (from step 10, page 14) at 750 nm (i.e., OD750). Note: For best performance, the OD750 of cultures should be greater than 1 and less than 2.

2. Harvest 1.5 mL of the cells (per transformation) by centrifugation at 14,000 rpm for 3 minutes at room temperature.

3. Remove the supernatant by pipetting.

4. Resuspend the cells in 1 mL of Gibco® BG-11 medium by gently pipetting up and down.

5. Centrifuge the cells at 14,000 rpm for 1 minute at room temperature, and remove the supernatant by pipetting.

6. Resuspend the cells in 100 µL of Gibco® BG-11 medium by gently pipetting up and down.

7. Add 100 ng of supercoiled plasmid DNA (i.e., pSyn_1 construct containing your gene of interest) into the resuspended cells. Mix the DNA-cell suspension gently by flicking the tube. In a separate tube, prepare a control transformation with the pSyn_2/Control Vector.

8. Incubate the cell-DNA mixture(s) in the 34°C water bath with a dark lid for 4 hours. After the incubation is complete, remove the tube(s) from the water bath and wipe them with 70% ethanol.

9. Plate 80 µL and 5 µL of each transformation mixture on separate BG-11 agar plates containing 10 µg/mL spectinomycin and pre-warmed to room temperature.

10. 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.

11. Incubate the plates for 5–7 days or until the colonies are ready to pick. Control vector should produce a minimum of 100 transformants per transformation. The results from the transformation with the pSyn_1 construct will depend on the nature of your gene of interest.

Then colonies were formed but I read some paper and it said SEGREGATION must be performed to get homogeneous chromosome since S.elongatus PCC 7942 has multiple number of chromosomes.

Do you have any idea how to perform segregation?

Does it take a long time?