Chloroplast Isolation from Chlamydomonas Reinhardtii

C. reinhardtii is a unicellular diflagellate green alga with a large cup-shaped chloroplast per cell. The presence of DNA in the chloroplast of this organism was first demonstrated by Sager and Ishida in 1963. C. reinhardtii is an organism well suited to study organelle-nuclear interactions because of its well-characterized genetics and the ability of researchers to transform either the chloroplast genome or the nuclear genome. In addition, C. reinhardtii can grow heterotrophically on acetate as well as photoautotrophically, which allows researchers to alter, delete and study genes necessary for photosynthesis. However, obtaining an intact chloroplast from C. reinhardtii is difficult because it accounts for about 40% of the total cell volume of C. reinhardtii and this large organelle is easily broken. Two previous strategies have been employed to isolate intact chloroplasts from C. reinhardtii. Both methods relied on cells that were cell-wall-deficient either through mutation (cell walls are absent or are produced in reduced quantities in cw-15 or cw-92 mutants as compared to wild-type cells1) or by treatment of the cells with autolysin, an enzyme released by C. reinhardtii during its mating sequence.

Fig. 1. The flow chart of chloroplast isolation from C. reinhardtii. (Yang M, et al., 2017)

Services

In vitro studies utilizing C. reinhardtii intact chloroplasts have been hampered by difficulties in purifying active intact chloroplasts with high yields from cells and mutants grown under different growth conditions. For many years, Lifeasible has been working on the independent isolation of chloroplasts from different plant tissues. We have successfully established a green microalgae model of Chlamydomonas reinhardtii to study many aspects of chloroplast lipid metabolism. On the one hand, we can provide the use of detergents or the use of pressure and shear to disrupt cells, yielding some intact chloroplasts, as well as disrupted cells and thylakoids. In addition, we provide a simple and rapid method to isolate intact chloroplasts from C. reinhardtii. The key to the method is to disrupt the cells with a fine needle and then purify from the homogenate using differential centrifugation followed by Percoll gradient centrifugation. Chloroplasts isolated by this method have been successfully used in uptake studies and other transport experiments. The basic process of C. reinhardtii chloroplast isolation is as follows:

(1) Algal culture: using synchronized lighting to keep all cells at the same size and growth stage, resulting in higher yields of intact chloroplasts.

(2) Cell disruption: using detergents, or pressure and shear, or narrow-bore syringe needles to disrupt cells.

(3) Chloroplast purification: gradient centrifugation purification.

(4) Quality assessment: quality was confirmed by electron microscopy, labeling enzyme analysis and ferricyanide exclusion test.

Competitive Advantages

• The chloroplast isolation procedure is fast, simple and reproducible, does not require specialized crushing equipment, and obtains high-quality, intact chloroplasts.
• This procedure can be completed within 3 hours and has been used to determine the localization of different transporters, as well as the localization of lipid biosynthesis proteins and the localization of accumulated PCBs.
• The implant retains its characteristic cup shape and is active in CO2 fixation.
• No pressure or detergent required to do it. Because no detergent is required, these chloroplasts are suitable for transport studies.

Lifeasible can meet the needs of customers on time and on budget through a wide range of chloroplast isolation strategies. Our aim is to be customer-centric and to provide the highest quality service to customers around the world. Our skilled and dedicated scientific researchers ensure that the most appropriate methods and techniques are selected for each specialized chloroplast project. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.

References

1. Yang M, Jiang J P, Xie X, et al. (2017) Chloroplasts isolation from Chlamydomonas reinhardtii under nitrogen stress[J]. Frontiers in plant science. 8: 1503.
2. Mason C B, Bricker T M, Moroney J V. (2006) A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii[J]. Nature protocols. 1(5): 2227-2230.