The Green Revolution aka GFP

In 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry “for the discovery and development of the green fluorescent protein (GFP)”. After its initial discovery in 1955 a lot of luck and hard work were necessary to push GFP to the forefront of a “green revolution” in biotechnology.  “Green” in this context does not refer to environmentally friendly or ecological, but rather to the actual colour: GFP is a protein (a fundamental building block of all living cells) that emits green light after excitation with blue light.

Why am I writing about GFP today? The protein and the revolution it has sparked are no longer really “topical issues” in biology, but GFP is so widely used that everyone knows about it and furthermore, I owe at least some of my interest in the life sciences to this remarkable protein.

Firstly, Osamu Shimomura discovered the protein in a species of jellyfish called Aequorea victoria, which, I feel, somehow bodes well. Secondly, the first time I ever wielded a micropipette or ran DNA on an electrophoresis gel was during a summer science camp at the Vienna Open Lab (http://www.openscience.or.at/#!/vol) in 2008. The one-week project was meant to introduce us rebellious and adolescent 15-year-olds to various molecular biology techniques, including microscopy, DNA restriction digestion and ligation, and polymerase chain reactions. The tutors taught these techniques by showing us how to engineer bacterial cells, Escherichia coli, so that they express GFP and would glow upon UV irradiation. Note that this was shortly before the announcement of the Nobel Prize that year. So here I am loading my first agarose gel (not entirely sure why I wasn’t wearing a left glove…). And the picture below shows the glowing bacterial cell pellet at the end of the week:

V + GelGreen E. coliIn and of itself this experiment was not useful (except that it relieved our burnt-out parents for a week) since fluorescing bacteria on their own don’t do much. However, the experimental techniques that we performed are standard procedures of molecular cloning (the manipulation – cutting/copying/pasting – of DNA, rather than making an exact copy of an organism).

Thirdly, when I entered the last year of secondary school I had the option of undertaking an “independent research project” and I chose to attempt one in Chemistry. The “thesis” – it’s really more like a long review article – was entitled: Green Fluorescent Protein – A Biochemical Perspective. I chose the topic because  of all the fascinating things out there in the science world, GFP was at least something I had already heard about/had some exposure to.

Lastly, since the start of my “real” research experiences I have worked with fluorescent proteins several times. Initially I thought that it was lucky to have landed in a project that utilises GFP, but in hindsight I realise that the fluorescent protein toolbox is so ubiquitous that it is difficult to evade. For example, last summer I was trying to find out where exactly within a fission yeast cell a certain protein was located: to do this I tagged this protein of interest with GFP (in practice this means fusing the gene encoding the protein of interest with the gene encoding GFP), which I could then visualise using a microscope. Wherever I saw a a green dot I assumed that the protein of interest was also present. To pinpoint the exact location of the protein of interest I needed to see a reference protein whose location was known. To this end we chose a protein called Mis6, which is found at a DNA structure called the centromere from where the two copies of DNA are pulled apart during cell division, and fused it to a red variant of GFP called mCherry. Now I could look down the microscope and simultaneously visualise green and red dots, and see where they were in relation to one another.

This summer GFP and I crossed paths yet again. The development of a new technology called CRISPR, which will be discussed in a separate post, is in the process of revolutionising molecular biology again, and its application in combination with fluorescent proteins is/will be extremely useful. In particular, the expression of fluorescent proteins inside cells (for example together with the expression of CRISPR components) allows machines to physically sort fluorescing cells from non-fluorescing cells in a process termed fluorescence-activated cell sorting. This in turn simplifies things for the researcher who can then only work with the fluorescent (i.e. biologically interesting) cells.

The above two examples only give a glimpse of how the fluorescent proteins are used in research today, but one other application that I find aesthetically pleasing is the so-called Brainbow, in which different types of neurons are labelled with different fluorescent proteins to yield images like this (from http://cbs.fas.harvard.edu/science/connectome-project/brainbow#):

brainbow2.1

The bottom line is that “seeing is believing” and a lot of science seems more convincing when we can actually look at an image of a (live) cell and convince ourselves of our hypotheses (or not, as the case may be). Not to say that there aren’t any caveats with using fluorescent proteins, including, for example, that some cells produce fluorescence of their own accord that may obscure exogenous expression of GFP.

Interestingly, although GFP and its cousins are now used routinely in virtually all molecular/cell biology labs around the world, the original function of the protein is still unknown. If you are interested in reading more about the “green revolution” then I recommend the popular science book “Glowing Genes” by Marc Zimmer or one of the many review articles available online (e.g. Kremers et al. (2011) Journal of Cell Science).

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3 thoughts on “The Green Revolution aka GFP

  1. Pingback: The Nobel Prize in Chemistry 2014 | Gotta Love Cells

  2. Pingback: PhD interviews – Round 2 | Gotta Love Cells

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