Tuesday, 31 August 2010

Disgusting Picture Finale (2)

It's a hard life, being a bacterial pellet. First you're brewed in a big set of flasks, before being spun into a solid mass and frozen at minue eighty degrees celcius for a number of days/weeks. Then you're thawed out at room temperature in some mildly pleasant (I assume) disruption buffer. If the term "disruption buffer" doesn't give you a warning, you're in for a big shock:

If you and your friends don't resuspend smoothly, you're targets for an old fashioned homogenising...

Once that's done, you have the essential appearance and texture of soup (lab hazard);

Until you've been run through the cell disrupter a few times:

Any researcher with a conscience would play Stabat Mater Dolorosa whilst this is happening, but usually the selfish fiends are more concerned with basic hygiene and the disrupter not overloading with pressure and exploding, sending shrapnel and bits of E. coli everywhere. Selfish.

After a 10,000g spin, this is what is left of the broken remains of our bacteria...nothing but a sulky grey mass in a gelationous supernatant.

So, add some Virkon (edible lab hazard) to it, and this is the result:

Once that has wreaked its work...

Ever want to spend a day in the life of a bacterial cell pellet? It's far from pretty...

Disgusting Picture Finale

You brayed, you demanded, you are probably nonexistant and I'm catering to my own self-indulgence. However the case may be, here are a series of pictures for your perusal. Don't say I ever bore you. :D

This is a CD machine. It essentially fires circularly polarised light at a sample and measures the extent to which it de-rotates it, which depends on the orientation of the alpha-carbon backbone present in proteins. It's vair vair complex, yet I don't feel I could do Dr. Dafforn's description of it justice. So here's a picture of the nitrogen tank that cools the mercury/halogen lamp:
It's very big.

Unfortunately, my sample showed little results here, so this is little more than "look at what I failed to get data from!".
Needless to say, it was disappointing, but circular dichroism is a technique I was intruiged by, and still am. It was fantastic to get a chance to learn how such a seemingly niche bit of machinery can be adapted for a similar yet important purpose.

Wednesday, 25 August 2010

Crashy crashy spin spin bang bang die

Today I tried my hand at ultracentrifugation. ANALYTICAL ultracentrifugation.
The difference between the two being that a normal ultracentrifuge spins samples at several hundred thousands of g's, whilst an analytical centrifuge does so whilst lasering the hell out of the samples. Check this out:
I'm sure many of you are mistakenly wondering why I've photographed a tumble drier. This thing costs about £250,000 and as such I wasn't allowed to operate it due to my lack of training. It costs labs £100-150 to use it and typically runs for over 12 hours straight, but the last use clocked in at 21 hours 24 minutes. Analytical ultracentrifuges can tell you all sorts of neat things (via lots of complicated maths) such as dimeric/trimeric etc. states, sedimentation coefficients, and whether your sample is in fact protein or, somehow, lard. I enjoyed a 2 hour demonstration by Rosemary, the lovable yet endearingly furious lab technician who was very enthusiastic about the whole peice of kit. Then I got to assemble the cell:

The sample goes into one of those holes, with some buffer as a reference. The transparent discs are made of sapphire, but the centrepeice is the expensive bit. The whole thing is worth three figures. I tried very hard not to drop it.

Sadly I misunderstood the necessary ingredients I would have to supply. "400 microlitres of sample and 500 of buffer" caused me to dilute my proteins almost out of existance and the signal was so weak a reprisal is necessary. Damn.

In other news, I added a mystery reagent to the mixture of membrane proteins we were binding to the resin overnight for the column elutions. Once bound to the column I pour the elution buffers through to get most of the useless proteins out before my protein washes out. However, this mystery reagent royally buggered up the binding mixture and I had to dilute it out by a factor of 1 in 4. So with two 60ml samples and a lot of precipitated gunk blocking the column, there was only one way I was going to finish before 6pm.

CAN YOU EVEN BEGIN TO UNDERSTAND WHAT YOU SEE HERE? Richard, in the other lab behind two sets of fire doors, had to sit down due to all the excitement in the department. Also he was doing lots of pipetting and his back would hurt otherwise. But seriously, despite the flow rate of one drop about every 10 seconds, it was unspeakably hectic.

I left the lab at about 6:20. Tomorrow I shall be doing Circular Dichroism with Dr Dafforn (hopefully), and shall be sure to attempt to avoid bursting the 5ft tank of liquid nitrogen in the process.

Thursday, 19 August 2010

Back in black

It's been a while! Mainly due to a nasty incident of pharyngitis which totally threw my routine. However, I took a mere two days off work whilst my supervisor was afflicted more than I by a similar condition, and an immuno-compromised friend I met for lunch on my first day back hasn't contacted me in a while. Anyway, in the three weeks since then, I've been doing all sorts of things, and in an insanely long update I hope to share with you the wonders of drippy things, defrosting, and GFP experiments.So first up, an update! I've made loads of bacteria, each single cell holding either lots of FtsA molecules, or ZipA molecules. They both do very similar things, but FtsA is marginally more enigmatic, useful and thus more fun! I'm working on ZipA. It's still quite cool, as both are of a similar size and have similar conserved regions (because those regions do very similar things) so it's essentially the same thing. In any case, Dr J and I have enough of these teeny bacteria to start messing around with them. If a bacterium has too much FtsA in it, and not enough FtsZ, which is the protein it latches onto to aid in division, it can't divide properly.

It starts to filament, as one bacterium tries to divide and fails miserably, and tries again later, and fails again, et cetera et cetera ad nauseum, until, BAM. Spaghetti E. coli.

See this? You can tell where the guys are dividing, but imagine they're reallly really long. May not sound too exciting, but when you're zoomed out so they look like carpet threads, and suddenly one starts to swim across the objective and just keeps going and going and going and going and going and going and going and going and going and going and going and going and going you can imagine how close I came to passing out and spending the rest of my life in some sort of rehabilitation centre for those who have seen what should never have been...not really, but it was very nice to see how the protein we've been overexpressing can drastically alter how this creature lives, and thus behaves. Imagine how multiple bacterial lengths, all communicating with each other and telling their partners to go different ways to find food, might live in our world. It's like siamese vigintuplets. :)
But enough of the science. Slapstick next.

I wandered into the lab a few weeks ago and almost collided with Dr H. Dr H has delivered many of my lectures in first and second year, she's an eccentric russian lecturer who started off in chemistry and sidestepped into biology. The woman adores NMR, which is a means of chemical analysis which pulls on all sorts of electronic and quantum strings, and is very helpful in terms of advice and finding reagents. Anyways, the first thing she told me was this:

"I'm going to cause a massive flood today!
"Me: "That sounds fun!"
Dr H: "Yes. See, this freezer needs defrosting and I've got this tray to catch the water!"
Dr H: *points to a large porcelain tray half in and half out of the freezer, teetering precariously on the brink of extinction*
Dr H: "Last time, at around 2am the water collected in the tray was enough to tip it over, and it smashed and water went everywhere!"
Me: "Why not just prop it up with something?"
Dr H: "...that's a great idea. Maybe I'll try that."

Please note the hazard-marked container propping up the attractive blue-rimmed china (?) tray. I feel this makes up for my destruction of a small measuring cylinder and a 10ml beaker. I swear as you stay in a lab longer your breakages become more catastrophic. A colleague left the autoclave on all day and someone else broke a £3000 column (it was easily recovered, thankfully). By the end of my career, maybe I'll devour Calcutta with a slipped keystroke. Virkon is powerful stuff, y'know.

So back to work! I've been taking membranes and cutting them into little polymer-encircled lipid discs, before purifying them to get rid of pointless things like lipids, lipid proteins which I don't care about, and Noel Edmonds, but it's been difficult. For one, the lipid discs are all the same size, and protein weight doesn't seem to have that much of an effect on the purification process. Also Noel continues to run amok.
However, our ZipA has a nice Histidine tag on it which allows us to search for it via affinity column elution. The general gist is, I mix the little discs of membrane with a pretty blue gel-like resin overnight on some sort of biochemical wheel of death, before plonking it in an open ended tube and pouring things through it. Changing the amount of salt in the things didn't work particularly well, but Dr J seemed to know how to mess around with the specifics and after upping the original salt concentration we started to play around with the imidazole concentration. Imidazole is basically similar to histidine, so it shunts the histidine tag that's bound to the resin out, causing our his-tagged protein to drop into the teeny plastic tubes we've placed underneath. We're cunning like that. Danny the champion of the world has nothing on us.

It seems that ZipA elutes (washes out) at around 25-50mM imidazole, which is useful as it's binding to the column (good) and coming out at a set value of elutant (good) and there's shedloads of it (very good) so if this continues I may be able to make some crystals and bring on the lasers! Well...Dr J and I may be able to go to Warwick, at which point Dr J shall ask them to bring on the lasers and I shall gawp and take pictures/notes like a beleagured first year.

But that's all I've done in the meantime; see, you've not missed THAT much. Apart from the chats with my colleagues, replication of techniques, and entertaining incidents in and out of that lab. Turns out Dr J is a massive Wing Chun Kung Fu fan, and has also bought what amounts to a steel-reinforced shed/bunker from eBay. As if I needed to sell summer projects to you much more. Just wait until my final report. :D

I'd feel amiss if I updated without sharing the last few days with you. Whoever you may be...

A guy from Hong Kong, via proxy of Warwick, came over to cut his membrane proteins into little discs. Accompanying him was his supervisor, who was the dude who gave the welcoming speech when I visited Warwick back in the day for my UCAS choices! He was as entertaining and interesting as ever, and it was a really nice chance to chat to someone from a different field of study who thought our technique was useful! Because it is, it is very useful.

Plus, so is he! As a master's student, the kid is very competent and seems eager to help out. When I told him what settings to use on our machines, he took down their reference numbers so he could convert the parameters to ones he could use on the setup down at Warwick. Plus he seemed much more resistant to caffine than I, and so could label eppendorfs (teeny plastic tubes as mentioned above) in a legible fashion. Coffee is bad. Very bad.

So today we did some purifications, and whilst Warwick's tagging method may not have worked, the protein was turning up in the flowthrough and in some of the early elutions, which is certainly a start. With a little fine-tuning, perhaps they'll get their protein out of the bacteria and into vitro, where it can be put to a very good use. Novel anti-peptidoglycan synthesis soon, perhaps! Or, in layman's terms, less bad bacteria, less of a need for yakult. I hate yakult.