Chlamydomonas, like a number of other kinds of algae, has a reversible hydrogenase that can reduce protons to molecular hydrogen. Melis et al. (Plant Physiology 122, 127-135, 2000) have shown that this activity is inducible in photosynthetically growing cells on deprivation of oxygen and sulfur. This process has potential economic importance as a source of hydrogen for fuel, especially if the native hydrogenase can be modified genetically to reduce its sensitivity to oxygen.
Hydrogen project kit for one student:
- Two Chlamydomonas cultures (CC-125) in liquid and one on agar plus stock solutions for 5 liters each of complete and sulfur-deficient media
- Copies of the Melis et al. paper and a review paper, plastic tubing, connectors and instructions
You will also need a fluorescent lamp and two 750 ml or 1-liter bottles of commercial spring water, preferably the kind with a pull-up drinking spout.
Hydrogen evolution supplement: Cultures of CC-125 (2 in liquid, 1 on agar) plus stock solutions for 5 liters each of complete and sulfur-deficient media.
10 ml liquid culture: 10 ml liquid culture of Chlamydomonas reinhardtii CC-125 for additional experiments.
Many Chlamydomonas mutants with impaired motility have been characterized. Because the structure of the Chlamydomonas flagellum is virtually identical to that of animal cilia, this system is an excellent model for investigation of flagellar and ciliary structure and function. Defects in central pair microtubules, dynein arms, radial spokes, and basal bodies produce characteristic alterations in swimming that are easily observed with a microscope.
Motility kit: a selection of motility mutants on agar, stock solutions and reference materials.
Chlamy Mating and Dikaryon Rescue
This is a laboratory demonstration of Chlamydomonas mating. Droplets of mating type plus and minus gamete suspensions are mixed on a microscope slide. Pairing and cell fusion occur rapidly, and are followed by a transient stage where the newly formed zygote swims about with four flagella. No equipment is required except microscopes, and it’s fun to watch. The quadriflagellate cell produced by gamete fusion remains motile for about two hours before the flagella are resorbed and formation of the zygospore wall begins. During this motile period, flagellar assembly continues, and polypeptides contributed by both parental gametes are incorporated into all four flagella. Thus mating of two non-allelic paralyzed mutants usually results in restoration of full motility in this transient dikaryon, as each partner supplies a wild-type copy of the defective flagellar protein produced by the other gamete. Watching the restoration of motility makes a good laboratory exercise for an undergraduate biology course.
Chlamy mating kit: The kit includes a selection of wild-type controls and complementing non-motile mutants for the dikaryon rescue experiment, plus instructions. Note: the major biological supply houses also sell Chlamydomonas mating kits for classroom use. We’ve been told ours are much more active.
*The mating and dikaryon rescue kit in liquid ready to use for 1-10 students should be used 24-48 hours after receipt as we induce gametogenesis the day we ship. Therefore, it is not recommended for users outside the US.
Phototaxis and Circadian Rhythm
Chlamydomonas cells perceive light using a primitive “eye”. The light receptor is a rhodopsin, similar to the visual pigment in human and animal eyes. A specialized cellular region beneath this light receptor, the orange “eyespot” or stigma, contains a layer of globules containing a carotenoid pigment. The carotenoid layer helps focus the light on the true receptor, the rhodopsin. Movement can be toward or away from a light source, depending on its intensity. Phototactic accumluation of cells can be demonstrated using just a petri dish partially covered with black electrical tape. A more elaborate apparatus for observation under a microscope, based on a design by Tony Moss, can also be constructed. These experiments can be extended to include analysis of several mutant strains that are unable to carry out efficient phototaxis because they lack the eyespot structure, are defective in the signal transduction pathway that enables the cells to respond to light, or can’t swim normally. Some strains of Chlamydomonas show a strong circadian rhythm of phototaxis. This can be demonstrated in cells entrained for a week on a light-dark cycle.
Phototaxis mutant kit: Cultures of wild-type and mutant strains in liquid for observation over a few days; no preparation of media or subculturing is required. For classroom use, we ship the cultures on agar and provide stock solutions for making the liquid medium. Some background material and references are also included.
Circadian rhythm kit: Wild-type strain CC-124 for the circadian rhythm experiment, plus stock solutions for culture medium, and instructions.
Combined phototaxis and circadian rhythm kit: To examine phototaxis and do the circadian rhythm experiment.
Nuclear mutations in Chlamydomonas segregate 2:2 in meiotic tetrads, whereas chloroplast mutations are transmitted only by the mating type plus parent in most zygotes, producing a 4:0 segregation pattern. Some years ago Jacqueline Hoffman at Washington University developed a laboratory exercise using chloroplast antibiotic mutations in both mating types that has proved to be a classic experiment for demonstrating uniparental inheritance in college genetics courses. Uniparental Inheritance kit: cultures of antibiotic resistant mutants, detailed instructions, antibiotics, stock solutions for culture media. If you’d prefer to provide your own antibiotics (streptomycin and erythromycin, available from Sigma, hospital pharmacies, or other suppliers), we can ship just the cultures and instructions.