Cyanobacterial Culture Synchronization and DNA Compaction Driven by Light-Dark Cycles

Abstract

Source: Murata, K. et. al., Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography. J. Vis. Exp. (2018)

This video demonstrates the synchronous cultivation of cyanobacteria under light-dark cycles to study circadian-regulated DNA compaction. Fluorescence microscopy reveals condensed DNA at defined time points, confirming cell cycle alignment across the population.

Protocol

1. Synchronous Culture of Cyanobacteria

1. Culture Synechococcus elongatus PCC 7942 on a sterilized blue-green medium 11 (BG 11) plate (in a 9 cm sterile plastic petri dish) containing 1.5% (w/v) agar and 0.3% (w/v) sodium thiosulfate.
2. Place the plates in a growth chamber at 23 °C with a light intensity of 50 µE/m²/s and subject them to 12 h light/12 h dark cycles.
3. Transfer the cells onto fresh BG11 agar plates once a week.
   NOTE: The cultures on the agar will appear as green bands of actively proliferating cells after one week under these culture conditions.
4. Take green clumps of cells with a flame-sterilized wire loop and streak the cells onto a fresh BG11 agar plate. Do this on a clean bench.

2. Monitoring by Fluorescence Microscopy

1. Use cells cultured on the agar plate for 6 days to observe DNA compaction. Collect cells from the plate at the end of the light period by pouring 1 mL of 0.2 M sucrose solution over the cells. Repeat pouring the solution onto the cells so that most of the cells are collected. Transfer the suspended-cell solution into a microtube for DNA staining.
2. Add DNA staining dye (e.g., Hoechst 33342) solution to 500 µL of the suspended cell solution in a microtube to a final concentration of 1 µg/mL. Then, keep the tube in the dark for 10 min.
3. Centrifuge for 1 min at 2,000 x g to sediment the cells. Discard the supernatant and add 10 µL of 0.2 M sucrose solution to obtain a dense cell suspension.
4. Transfer 1 µL of the solution containing stained cells to a slide glass, put a cover slip, and observe with a fluorescence microscope equipped with an ultraviolet (UV) filter using an objective lens with a magnification of 100X and immersion oil.
5. Confirm that the DNA compaction is observed at this point in most cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hoechst 33342 solution	Dojindo	346-07951	1mg/mL in water
Agar powder (for plant culture)	Wako	016-11875
Boric acid	Wako	027-02192
Manganese chloride tetrahydrate	Wako	139-00722
Zinc sulfate heptahydrate	Wako	264-00405
Sodium molybdate	Wako	196-02472
Copper sulfate pentahydrate	Wako	039-04412
Cobalt nitrate hexahydrate	Wako	031-03752
Sodium nitrate	Wako	191-02542
Magnesium sulfate heptahydrate	Wako	131-00405
Calcium chloride dehydrate	Wako	031-00435
Citric acid	Wako	036-05522
EDTA-2Na	Dojindo	343-01861
Sodium carbonate	Wako	197-01581
Potassium phosphate dibasic	Wako	164-04295
TES (Good’s buffer)	Dojindo	344-02653
Ferric ammonium citrate	Wako	092-00802
Sodium thiosulfate pentahydrate	Wako	197-03585
Fluorescent microscope	Nikon	ECLIPSE 50i