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.

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.

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.

Two at a time

Just thought I'd show you this: it's a heat block! I have to break apart liquid cultures so their contents will leak out and be detected in the SDS-PAGEs I do. So I usually set it to 98 degrees and cook them for 10 minutes. I don't see the point in the warning on the top though..."Warning - the block may be hot!"

It's a heating block. That is the absolute MINIMUM I'd expect.
So last week!
I performed a western blot on all the cultures we'd grown in order to make some FtsA recently. The small-scale cultures were in a nice little lunchbox-style array with several wells and a squidgy silicone lid. However, there were 16 wells with bacteria living in them, and one SDS-PAGE can only hold 8 lanes, as two have to be reserved for protein markers, which we can use to compare the bands we get to bands of defined mass!
So I had to do two at the same time. It was intense.




You can't tell very easily because the bubbles produced when the power's turned on are obscuring it, but THERE ARE TWO GELS IN THERE. It's also nice to check back occasionally on the apparatus to make sure I've connected the electrodes the right way round, and that the band isn't currently travelling upwards, which would be bad.


So after that had run, I took out the gels, sidled them with a nitrocellulose membrane, wrapped them between sponges and filter paper, and stuck them into the buffer. But unfortunately whilst I was otherwise engaged I forgot that I'd run two at once. So when I opened the lid after an hour I saw this:


Me: "OH IN THE NAME OF EVERYTHING THAT IS HOLY, THERE'RE TWO OF THEM!"

I almost soiled myself. However, composure and the ability to cope with previously unencountered phenomena can make or break a scientist, so I calmed down and adapted, excising the membranes and placing them in blocking buffer, antibody, SDS-Tween and then another set of buffer and antibody:



THERE ARE TWO GOING AT ONCE! CAN YOU EVEN BELIEVE?

Ultimately it was a challenge in terms of organisation and dexterity, but it worked well ish. The antibody we used was new and may have had some teething problems. However, despite this there was a signal present, albeit a rather weird one. So I'm running a comassie stain which will hopefully shed some more light on this quandary.

Oh and also I got to mess around with some liquid nitrogen, but that's really boring, so I didn't take any pictures.

When the going gets tough, the tough steal an obselete cooling device from another room and shove it between the wall and a disrupter

Pretty, isn't it? I think it goes well with the tubing.
This is an FC-360 cooler thingy, and as its instructions were not on the supplier's website Dr J and I had to guess, based on a later model, which buttons would be less likely to result in a smelly inferno. It worked surprisingly well, cooling the lysed cells down to 0.5 degrees celcius. Oh, yes...the cells...

Look at them! All frozen and such. To thaw them, we suspended them in breaking buffer, a cocktail of tris and other such lovely chemicals. Then we stirred them for some time until it held the consistency and appearance of mushroom soup (deceptive yet inedible lab hazard). Then protease inhibitors were added to prevent the cells from digesting themselves once lysed. Unfortunately these turned the goo into what resembled crab soup in colour and smell (deceptive yet inedible lab hazard). It stunk most of the lab out rather quickly. Nasty stuff.

Anyways, the disrupter was pretty effective, turning the mush of defrosted bacteria into a thick soup of cellular contents and fragmented membranes. As it was only the membranes we're interested in, they were spun at 10,000rpm for 30 minutes, and then 35,000rpm for an hour. After removing the supernatant, what remained looked a little like what you get if you leave a red fruit pastille in your mouth until it's a bare slither betwixt your tongue and your palate. Unfortunately it still reeked of crab soup (deceptive yet inedible lab hazard).

Whilst the membranes were being pelleted in a snazzy new machine with a delightfully retro interface (think the old school thumb-scrolled wheels on ancient white-numbers-on-black-squares alarm clocks), Dr J showed me how to set up a load of yeast wells for culturing. Tomorrow, I'll be taking the optical density, doing some crazy (simple) maths to work out how much to transfer into the new set of wells, and brewing up a set for western blot analysis. I'm sure it'll be a lot of fun, and hopefully I'll not grow anything lethal by accident. Although a Zombie Apocalypse would make for a great report, I doubt the novelty would last.

I'd give it about 28 minutes.

Nobody's Perfect

I've not updated recently, and that's mainly because a lot of the technique I've been using has been repeated over the past few days. In order to get enough protein to survive the purification etc. stages, a lot of bacteria is needed; thus I get to do lots of incubations and the such. Whilst I'm getting pretty good at it (for a second week student, anyone else here could probably wipe the floor with me), I do make mistakes. Here are some of them:

(After making a western blot)
Me: "Hmmm. This doesn't look right at all. It's...rather smeary. And I swear the band's moved down a lot...I understand as my technique isn't fantastic the line might be rather thick, but it should be in roughly the same place. Maybe the exposure was too high? No, that affects signal...argh, something's gone horribly horribly wrong. Dr J, help!"
Dr J: "Nah, it's fine. You've just got it upside down."

Then there was that time I accidentally forgot to put ampicillin into the flasks. And the time I didn't clean the virkon (bleach) out properly and incubation took SEVEN HOURS.

But ultimately these mistakes are helping me learn what not to repeat.

In other news, time to outline my project, as that's not been done yet.

Baaaaasically I'm looking at ZipA, primarily, whilst doing a little bit of work with Dr J on FtsA which is similar in that it also binds to the cell membrane and to FtsZ. The first stage is to make lots of it, the second is to purify the protein (which will result in much loss, hence the requirement for so much bacteria). After that we can do some tests on it, including perhaps crystalisation, which will be neato.

"So how's the project going?" I ask myself in some sort of bizarre self-interrogation. Well, well. The best part is seeing myself get better over time at little aspects of the techniques, whilst the worst part is the sinking feeling of dread as alarms sound and the order is given to "evacuate the facility, it's loose, it's loose". Fortunately these incidents are mostly dreams and actually everything's going as smoothly as could be expected. Next week may be a large departure from the work I've done so far and I'll be sure to write lots and lots about the fun new things I've learned how to do with machines and my brain.