CAPP

Combining Algal and Plant Photosynthesis

Experimental Techniques

On this page you can (or will be able to!) find information about the kinds of scientific techniques we use in the course of our research. This is intended to inform young students, perhaps soon embarking on a scientific degree, or already on one, of the techniques that are available to scientists and how they can be applied to real research.

Microscopy – Light, Confocal, Electron

Old-school observations are essential for observing the presence or absence of a pyrenoid in various mutant and wild-type (WT) Chlamydomonas strains, as well as for simply counting our Chlamydomonas cells in our liquid cultures so that we know how many we have. Depending on the level of detail in which we need to observe our cells, there are various types of microscopy we may use: for example, light microscopy allows us to view whole live cells very quickly and easily, and confocal microscopy can allow us to visualise, for example, fluorescently labelled algal cells, while for more detailed intracellular images, the cells must be dehydrated, fixed, sectioned and stained, and viewed using an electron microscope. This allows us to see details like starch plates around the pyrenoid, the Golgi apparatus, and sometimes perfect cross sections of a Chlamydomonas flagellum.

          Fullscreen capture 10072013 114401.bmp           Images taken using light microscopy (left) and transmission electron microscopy (right)

Mutagenesis

One of the most indispensible tools in modern molecular and cellular biology is the ability to genetically modify, or “mutagenise”, our study organisms, and observe the resulting changes. In the context of our studies of the Chlamydomonas pyrenoid, mutagenesis is crucial for creating hybrid RuBisCOs – that is, RuBisCO molecules with a combination of genetic sequences from Chlamydomonas, which has a pyrenoid, and, for example, spinach, which lacks a pyrenoid. This is an important technique for discovering the genetic basis of pyrenoid formation, which will be crucial if we are ever to engineer the algal CCM into plant cells. We also rely on mutagenesis to probe other elements of the algal CCM. For example, we can artificially “knock-out” candidate transporter or carbonic anhydrase genes, and observe the resulting phenotype, in order to confirm their function.

Fullscreen capture 10072013 111203.bmpWildtype Chlamydomonas has a characteristic pyrenoid, but the hybrid RuBisCO – Chlamydomonas large subunits with spinach small subunits – expressed in Chlamydomonas results in lack of the pyrenoid.

GFP-tagging

There are a number of methods by which we can determine where in the cell certain gene products are localised. One such method is to engineer into the cell’s DNA a genetic construct in which the gene of interest (often shortened to GoI) is fused to an adjacent gene that has a conspicuous gene product that can be easily visualised. One such gene is that encoding Green Fluorescent Protein, which is from jellyfish. By inserting a construct into a Chlamydomonas cell that contains, for example, a candidate transporter gene fused to the GFP gene, a fusion protein should be produced. The protein should be localised as would the ordinary transporter gene, but the GFP tag attached to it allows us to see where this is to. This investigation is currently taking place.

Immunogold-labelling

Immuno

Another method enabling visualisation is immunogold-labelling; this operates by using a specific primary antibody to bind to the Chlamydomonas RuBisCO and subsequently adding a colloidal gold nanoparticle-bound secondary antibody. Thus the cell’s RuBisCO can be located by observing black dots whereby the electron-dense nanoparticles scatter electrons in transmission electron microscopy (TEM).

Rubisco immunogold-labelled pyrenoid

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