A blogger (Black Knight) left a comment on my blog yesterday and in an idle moment I went to see what sort of things they were interested in and found:
This has got to be in the running for the coolest cloning experiment ever.Last Tuesday a grad student in the reciprocal space cadet lab, let's call him Fu Manchu, asked me if I had any GFP. 'GFP' expands to 'green fluorescent protein', which is a protein that is green, and, um, fluorescent. It's naturally found in a certain jellyfish and glows green when you shine a certain wavelength of blue light at it. This is really useful for studying where proteins go inside cells or whole animals, because you can join the DNA that codes for GFP to the bit of DNA that codes for the protein you're interested in, and put that into your experimental model. Much like I did here, there and elsewhere, in fact.
But Fu Manchu didn't want to localize a protein in a cell (because as I hinted above, he's a scatter-brain and wouldn't recognize a whole cell if it mitosed); he wanted the protein itself to use as a crowding agent in some unspeakable experiment. I had to tell him that no, it wasn't the sort of thing I kept in my fridge, but I did have the DNA that codes for GFP in a plasmid vector and he was quite welcome to take it out and make the protein in a system of his choice. It would take a few days to design and make the primers and do the PCR but it would be simple enough.
I went to have a cup of tea and realized that actually, GFP in a protein expression vector, that is, in plasmid that we use to make vast quantities of purified protein rather than in a different sort of plasmid that we use to make relatively small amounts of protein in situ, might be a useful reagent and I was a damned fool for leaving a very similar reagent in Cambridge and not getting it shipped over with my other useful bits and bobs (and I couldn't get it shipped in the time frame that Fu Manchu wanted it). So I trooped back to the computer and had a look at restriction sites, and realized that if I was only semi-clever I could cut the GFP gene out of the one plasmid and into the other sort.
So on Wednesday I set up a couple of enzyme digests, purified the appropriate bits of linearized DNA and set up the reaction to stick that gene for GFP into this expression vector, where it would, if I did nothing else, make GFP and GFP alone (and left a couple of restriction sites so that I could, at a future date, drop in the gene for some other protein after the gene for GFP and make green some-other-protein).
To retrieve the construct made in this way I had to transform some bacteria — in other words, persuade them to take up this new construct and propagate it. The bacteria can be persuaded to do this because the vector you make has a gene for resistance to some antibiotic on it, and you select only the bugs that contain the plasmid you want by growing the bugs on plates that contain that antibiotic. Thursday morning, then, I hoped to see colonies on my plates; each colony having grown from a single antibiotic-resistant bacterium.
That's the theory: in practice you always get 'background'; bacteria that grow because they have taken up some plasmid that doesn't have the gene you really want, or one of a myriad other excuses. And you then have to make DNA from several of these colonies and cut them up in special ways and all sorts of tedious stuff. Knowing this, I chose the bacteria that I would transform to be the sort that cannot help but make protein, even when you don't want them to. And I reasoned that the bacteria that had the right plasmid, that is with the GFP in it, would be green.
On Thursday morning then, I took my plates upstairs — which indeed had colonies — and asked to borrow Tiffany Case's fluorescent microscope. I told her what the plan was, and we looked at the plates together, and this is what the butler saw:
That's two photographs of the plate. On the left, normal light, and you can see all the colonies. On the right, we've illuminated with blue light and the only colonies you can see are the ones that are making GFP, and are therefore glowing green. Ignore the black pen marks — they're just from when I counted colonies. Here's a closer look:
See those two really bright suckers? They're making GFP from the DNA I gave them. That third colony isn't, and therefore glows not. So on Thursday afternoon, forty-eight hours after conceiving the experiment, I was able to hand Fu Manchu a fresh plate containing bugs that make the protein he was after.
See those two really bright suckers? They're making GFP from the DNA I gave them. That third colony isn't, and therefore glows not. So on Thursday afternoon, forty-eight hours after conceiving the experiment, I was able to hand Fu Manchu a fresh plate containing bugs that make the protein he was after.Basically, at this moment you're either going"Dude, that is so totally awesome!"
There is no middle ground.