This article details a method for isolating plastoglobules from cyanobacteria, specifically Synechocystis species PCC 6802. The process involves cell disruption, density gradient centrifugation, and careful harvesting of the plastoglobule fraction for further analysis.
Begin with a culture of cyanobacteria, which are photosynthetic bacteria.
These bacteria contain lipid-rich plastoglobule droplets and thylakoid membranes, which are folded membranous structures rich in photosynthetic pigments.
Centrifuge the culture and discard the supernatant to remove bacteria-produced extracellular polysaccharides. Wash the pellet, then resuspend it in a buffer.
Use a high-pressure homogenizer to generate shear force, which disrupts cell walls and membranes.
Rupture of the thylakoid membranes releases photosynthetic pigments into the lysate. The pigment release causes a color change that indicates cell disruption and plastoglobule release.
Load the lysate into centrifuge tubes, then add a sucrose solution to form a density gradient.
Centrifuge to separate cellular components by density.
The thylakoid and other heavier components pellet at the bottom, while the low-density plastoglobules float to the top as a distinct band.
Collect the plastoglobule fraction and store it by freezing for further analysis.
Grow 50 milliliters of synechocystis species PCC 6802 culture in BG11 media for 7 to 10 days to reach the stationary phase.
Using a spectrophotometer, adjust the cell density to an optical density of 2.0 at 750 nanometers. To remove the polysaccharides, centrifuge 50 milliliters of culture and remove the supernatant. Repeat washing of the cells in 50 milliliters of buffer A two times.
Resuspend the washed pellet in 25 milliliters of buffer A and break the cells using a French pressure cell at 1,100 pound square inches. Repeat the process three times in cold conditions until the lysed color changes from green to red-blue-green under white light. Remove the aliquot of the cell homogenate and set it aside to be stored as a representative total cell sample.
Distribute the resulting homogenate in eight three-milliliter ultracentrifuge tubes filled to a maximum of 2.5 milliliters in each tube. Overlay this homogenate with 400 microliters of medium R, producing a step gradient. Carefully balance the tubes by adding additional medium R as necessary before centrifuging the tubes.
The thylakoid and other heavier organelles, including any polyhydroxyalkanoate bodies, show pellet formation, while plastoglobules form a yellow oily pad on or near the top of the sucrose gradient. Harvest the resulting floating pad of crude plastoglobules with a syringe and deposit the harvest into a two-milliliter tube.
Scrape plastoglobules off the side of the ultracentrifuge tube wall with a needle tip if necessary. Either continue with cyanobacteria processing or store crude plastoglobules at minus 80 degrees Celsius and purify later.
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