They’re pale and creamy, scattered across the agar like diseased lesions. Something about the three-dimensionality of the colonies – vaguely dome-shaped instead of flat – rings some sort of primordial alarm bell deep in my hindbrain. Although I know it’s irrational, I can’t control the little shiver of revulsion each time I stab one of the glistening disks with a sterile toothpick and spread it over the next pie-shaped zone of the fresh plate. Even the way these yeast people have taught me to streak seems inherently wrong: my Microbiology tutors at university would be horrified by the laissez-faire way I am now scribbling onto the surface of the agar, with no system or intent to isolate single colonies.
Of course I am thrilled that all of my transformations yielded so many resistant colonies, but to this biomedical researcher, yeast are only ever bad news: they infest your tissue culture cells, grow inappropriately on your bacterial plates and cause all manner of nasty infections in patients.
So, after months of sitting in front of a computer, I’m finally back at the bench. Just today I hit the shiny ‘submit’ button that should consign my further revised manuscript to its final acceptance (seeing as how the editor had only asked for minor text changes); and after a visit from my collaborator last week, it was clear I couldn’t proceed further on data analysis until he’d performed a few more bioinformatical dark arts with our dataset. All this meant that I was free to escape to something more interesting in the interim.
So escape I did, to my lovely new collaboration with the local Schizosaccharomyces pombe guys down on the first floor. Even after a few days with this crew, I am starting to suspect that fission yeast people are inherently cooler than your average biologist. For starters, they listen to music in blatant disregard to institute rules – not loud enough to trouble anyone, but just loud enough to give off a rebellious vibe. And of course, the entire place smells faintly of beer.
It’s a brave new world: I’ve just made a series of mutants of the pombe homologue of one of the most promising novel hits from my cell shape screen. In one week, you can knock out your gene; you can replace it with a tagged version of your gene under its endogenous promoter; and you can put in a version of your gene under an inducible promoter, all down to the magic of recombination. We made monster recombination cassettes using 100-mer PCR primers and then just zapped them into the bugs – the pombe did the rest. I estimate that it’s taken me ten days to achieve something that would have taken two to three years in a mouse.
It’s already exciting: I can see that my deletion mutant transformant colonies are noticeably smaller than the wild-type, so there might be some sort of effect on growth. In a few days I’ll be able to peer down a microscope and visualize the tagged protein to see where it is localized in the cell, and I’ll be able to look at the knockout and overexpressing cells in more detail to see if I can discern the nature of the defect. Of course once I have a clue about pathways, there is an entire genomic library of yeast strains that I can mate my new strains with, to look for genetic interaction all in a matter of a few days.
And the best bit? By convention, I get to name these new yeast strains after my own initials, and they will pass forever into the global community’s strain collection. JLR-1, -2, -3 and -4 may not be as exciting or poetic as a Drosophila gene name, but it’s a hell of a lot more personal.
Hey! Structural biologists also listen to music you know!
At least, in labs where we’re serious about getting crystals (thinks: that’s probably why my Sydney projects were so pants–strictly ‘no music’.)
What I like is that since there is music, nobody is wearing iPods, so people actually chat. I’ve really missed this, so I’m glad to be hanging out in a different lab now and again.
Ah, I do like me some yeast genetics… my experience with the much less exotic cerevisiae rather than your pombe, though.
The coolest thing IMHO (apart from red/white colour selection, which is fabulous) is the ability to separate all the chromosomes on a gel. Ok, a pulsed-field gel to be sure, but a gel. Lots of fun.
If you’re really missing streaking to single colonies, you could always try to evaluate your mutants’ growth potential by doing limiting dilutions and counting colonies (and thus cells at a time point). You probably thought of that and decided against it already… 😉
I am so excited, I just looked at my GFP-tagged mutant under the scope and it’s green! Pombe is absolutely gorgeous – sexy septa to die for.
I think nowadays they use a FACS machine to assess growth – boring but serviceable. Personally, I’m looking forward to my first phalloidin stain, because I suspect my pet gene will disrupt actin patches.
We demand pictures!
And good looking for differences in yeast cytoskeleton. Them little buggers is so small I find it difficult to tell what’s going on.
I’ll take some pics tomorrow. Meanwhile still swooning from ye olde fashioned phenol extraction I just had to do on the yeast to isolate genomic DNA. Makes me misty-eyed for the TBQ (Time Before Qiagen). Them cell walls are some tough mo-fo’s.
Can’t beat phenol. More, I say, more. And if it kills some grad students, well, we have a funding crisis don’t we?
I totally understand! I recently started working with yeast in a cancer biology lab b/c my tissue culture work was taking forever and I love speedy yeast. The whole open lab (three different labs) turned on me like I was diseased. I got a lot of “keep your yeast away from my cells!” Geez!
I’m getting the opposite reaction from the yeast lab – they know I work in a fly lab and are terrified that the budding yeast used to feed the Drosophila might contaminate their pombe.
It’s all a matter of perspective…
and so began the Yeast Wars of ’10…
Lauren’s message rang an instant bell. I have worked with a yeast-like organism (albeit a pathogen). I love them. They are docile. They sit on the plate, don’t go anywhere, don’t jump around and aerosolize on their own and creep into nooks and crevices and other people’s cultures – as do molds. Therefore, I was mighty surprised when the institutional animal facility (of my university) raised a ruckus about my using ‘flying yeasts’ (!!) in a biosafety cabinet in the shared animal room.
Flying yeasts? #awesomesauce
Kausik, you never should have let them use the jet packs. You have only yourself to blame.
But seriously, I’d never allow yeast cultures in my cell culture room, let alone hood or incubator. They may not fly, but they are pretty good at wafting around, especially during the hot summer months.
Or am I just yeastist?
Jennifer,
I have to admit your post title reminded me of the Lablit blurb for Paul Brand’s novel…!
(Saying nothing about you personally, it’s just I had only just read and written about that not so long ago, then saw your title!)
Isn’t the genus name Drosophila getting replaced?
Well. the postdoc who slept with the enemy in that book didn’t know he was a baddie until it was too late! When it comes to yeast, I’ve got my eyes wide open.
Don’t know about the fly genus nomenclature – I’m only a Drosophila poser, you know…
Phenol? PHENOL?
You is heartless cruel you is.
I seem to remember digesting away yeast cell walls to make spheroplasts using… um… er… zymolyase perhaps? This was to make PFGE-quality HMW DNA (agh! abbreviation salad!), so ruckusing them with phenol and vortexing and suchlike wasn’t an option.
I am this: old.
Guest comment; Jenny can’t comment from her iPhone but I, apparently, can. This is what she says:
Phenol, because I’m just doing a PCR – I was shocked that I couldn’t just swish the colony in PCR buffer, though. Today I had to lyse them for proteins – it was total carnage. My hands are still faintly vibrating from the excessive vortexing.
Jennifer,
Oh, absolutely! No yeasts in my cell culture room either… But I was talking about a Type II biosafety cabinet, in our mouse room, inside which we (and other people) do the infections. And hello! Our yeasts (Candida and Cryptococcus) don’t waft… They have been well-trained and plate-broken. Honest.
Jenny> I discovered some nice new tubes that have a little “plug” in them to help separate phenol and chlorophorm from the “things you want” and reduces the smell and increases the separation a lot. If you are interested I’d send you an email with the specifics? I loved them, so much easier and people in the lab didn’t hate me when I pulled the phenol to the hood 😉
My hands are still faintly vibrating from the excessive vortexing
Ah, I remember that feeling from mushing up C. elegans by vortexing with glass beads.
Hm, what was I saying about “heartless cruel”?
Am ridiculously nervous today, about to find out if any of my recombinants are bona fide. At least my genomic DNA preps had pellets – I must still have that phenol touch.
and…?
We’re all waiting with bated breath, here.
Apparently yeast colonies are surrounded by dead wild-type cells that contribute to genomic DNA…so I’m still try to work out what is background and what is real!
Sequence it. You know you want to.
I haven’t sequenced genomic DNA since I was in grad school, so things might have moved on, but there’s no way to do it without cloning it, is there? They aren’t plasmids – these cassettes are integrated into the genome. Is there a way to do it without cloning? Don’t fancy making 60 subclones.
p.s. It’s not looking good, but I think I know why.
CLONE it?
Surely you jest.
PCR the cassette and cycle-sequence the amplicon. Done. Dusted.
Winty’s da man when it comes to sequencing.
Protocol pleez?
I’ve asked our sequencing boffins to send one along (to me, and thence to you). In the meantime though – isn’t this as simple as PCRing your cassette out of the genomic DNA and then sending the resulting PCR product along to your friendly neighbourhood sequencing core facility (perhaps with a primer or two)? Am I missing an important logical step?
No, you’re talking to someone who hasn’t sequenced genomic DNA since 1995. I was a whizz at M13 cloning – after 6 years and over a megabase of manually sequenced (35S dideoxy) DNA, I could pick just one plaque and it would be guaranteed to have insert. Only way to sequence then. Now, I’m sure it’s easy – protocol appreciated!
p.s. I wonder how clean the DNA has to be? The yeast protocol is pretty crude – phenol with no chloroform clean-up, OD 260/280 a meager 1.7, &cetera.