Sunday, 25 July 2010
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.
Monday, 19 July 2010
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.
Friday, 16 July 2010
(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.
Monday, 12 July 2010
I'd also made one last Friday as well.
In chronological order, last Friday when I cleaned out the flasks I didn't rinse them out sufficiently, meaning there was some residual Virkon (raspberry sherberty bleach stuff) in all 5. This stopped the bacteria from growing quite as well as they usually would (which is fortunate in one way; bleach that allowed or potentiated bacterial growth would be both useless and still not delicious). That was a bit of a bummer, and I read their optical density (OD) at 895nm to check that they weren't as numerous as I'd hoped. Dr J posited that we leave all but one of the flasks in the cold room, and if there were none in the solitary flask the next day we could re-inoculate. I left them for another three hours before reading their OD again; this time they definitely smelt of bacteria, and they were pretty cloudy and warm, but the OD was still pretty low. Subsequent investigation revealed I should have been reading the absorbance at 595nm, which makes a lot more sense. Turns out we have more than enough cells, so I inducted and slammed them back into the incubator overnight. It's a pretty simple mistake and I'm annoyed that it stopped me from doing more with today, but it's not one I'm going to repeat.
Lookit my flasks! Clearly full of microscopic protein factories.
As a brief digression, I think it's time I talked about gloves.
The gloves I've been using are totally kick-ass. They're dry nitrile gloves (like the ones in the chemistry laboratories) rather than the awful loose powdery ones the 1st and 2nd year bioscience students get; but unlike the chemistry gloves these ones are purple. Purple, asides black and possibly neon green, is the best colour for gloves. Unfortunately, they are *dry* nitrile gloves, so if hands are wet (say from something as innocuous as a recent handwash), they're almost impossible to don. Here is a visual illustration of today's struggle:
Getting the glove onto the hand can be tricky, but once on it's usually just a matter of working through finger by finger.
Success! This took about a minute and a half. Now for the other hand!
Friday, 9 July 2010
So first of all I made the medium for the cultures, like I said. Theeeen I inoculated the flasks I made up with some bacteria, and lowered the inductant concentration to 0.25M. Dr J said this was probably going to give similar results, so it makes sense to not use too much of the stuff. After a series of optical density readings to test how much bacteria there was (not too much) we left them to incubate overnight.
The next day there were lots of bacteria. Lots and lots. Some might say too many bacteria. But fortunately they'd be wrong. We spun the cultures down and collected them. Here are some pictures of the bacterial pellets:
And here's one of the total mass we collected...23.4g! (give or take the weight of the bag. Also viewable are Dr J's hand and the sun, making a surprise appearance
Looks a lot like a heart, or an upside down arabian peninsula. Either way we were well on our way to making a stromatolite.
After collecting these...horribly malodorous creatures, I processed two samples I'd taken from the culture before spinning it down. To do this I made my own gel which was oddly relaxing, until half the lab called dibs on my spare gel and I had to wrap it in foil before hiding it in the cold room behind some agar plates.
Awww, they're like my little gelatinous babies. Although if I ever do have children, I will probably never inject protein markers and buffer-suspended bacterial contents into them before immersing them in chemicals and running an electric current through their tank. Probably.
After this I performed the western blot as before, taking care not to quench my thirst on either the milk blocking buffer or the concerningly named "anti-mouse HRP", which sounds a lot like some sort of niche pest-control surface-to-surface missile launcher.
Eventually we processed the result with hydrogen peroxide and luminol solutions before taking some more pretty pictures of it (which are forthcoming). Dr J was very pleased with the result because despite reducing the inductant concentration the signal was still at least as strong as his assay, and although it was impossible to tell whether we'd get more protein from overnight incubations (as we used the same sample volume both times ergo rougly similar amounts of protein in western blot sample) we definitely had over twice the biomass of the previous assay, meaning potentially lots more protein. I was pleased with the result because nothing had caught fire over the previous 3 days, the camera hadn't exploded, and nobody in the lab had any form of chemical burn.
So next week will see me doing as many inductions and incubations as possible to get lots of bacteria for the crucial and exciting purification and analysis steps, which may involve crystallography, a real "proof of concept" exercise which I'll talk about some other time. My personal target is to get the mass up to 100g-ish, which should take about 3 more incubations.
How did I know how to do all of this? I don't have amazing memory, so I've been taking notes like crazy, which I then write up so I have a neat set of instructions, and a pad on which I write down what I've done and when so as to keep track of my movements and any mistakes I may recognise later. Don't believe me? See for yourself:
Wednesday, 7 July 2010
On arriving at the lab the membrane was retrieved from the cold room where it had rested in milk overnight, and we continued the washing with antibodies. As each wash took an hour I did some reading about yeast two-hybrid systems, which seem to be a pretty neat means of testing for the expression of two proteins which can interact with each other by affixing various gene activation and DNA binding domains to them. While I doubt I'll be using that in this lab it helped me understand a little more of these sheaths of papers I'm totally reading instead of going on facebook and playing endless spider solitaire.
On a related note, as well as reading up on FtsA I've been chatting to the post-grads and masters students who share working space with the Dafforn lab on occasion. As well as being really good eggs they're also working on some pretty interesting things, such as testing whether a protein is involved in oxidative stress responses by bleaching the hell out of bacteria with some peroxide. I'm pretty certain a post-grad course is for me.
Once the membrane was suitably covered in milk and protein, we turned the camera and controlling laptop on in the dark room. Here is a picture of the camera:
I've got to be honest, it does look a little bit like a coffee machine, only with more radiation hazard stickers. The lens is up at the top, and if you open the red door there's an adjustable tray on which you put your sample. An initially disconcerting number of switches on the front contain white light, UV, Power, and other such useful functions, whilst different lenses are available for different settings. We were using no lens and basic white light, which is good, as those UV-opaque faceguards looked uncomfortable.
We also made up a solution containing hydrogen peroxide which interacts with horseradish peroxidase to produce a signal detectable by the camera. After dumping this onto the membrane for 2.5 minutes Dr J transferred the membrane to another sheet of clingfilm and placed it into the camera's chamber, before taking several rather clear pictures which I shall show you later on. In the meantime, here's a picture of a tornado:
This is me making up a fantastic four litres of liquid broth, which will be used to grow bacteria in. I added 80g of the stuff and then the water, although really I should have added the flea first, as this is the bit that is responsible for the stirring. Several attacks with a long plastic rod later it was whirring around at the bottom merrily as an alarmingly deep whirlpool emerged. I had a while, so I took the above picture. All I could have added were two Galleons and the soundtrack to Pirates of the Carribean 3...
Embarassingly I mistook the foam at the tip of the funnel for some undissolved LB and Dr J had to turn the stirrer off to demonstrate this was not the case. I shall have to guard against being hypnotised by swirling liquids.
Once the broth was made I decanted it into 5 flasks and bunged them up, ready to be autoclaved. Nothing sterilises liquid broth quite like a good autoclaving, apparantly. I'd imagine a pressure cooker the size of a laundrette drum would sterilise most things.
Ultimately it was a pretty productive day; Dr J got a nice set of photographs showing his protein is being expressed nicely in his recombinant E. coli, and I got to make a storm in a 5-litre beaker. Not to mention the training for when I do all that myself.
Tuesday, 6 July 2010
And by excitement I mean Western Blotting.
I love jelly, so I was right at home with the SDS-PAGE (PolyAcrylamide Gel Electrophoresis). After making up the gel with such wonders as TEMED (smells like fish), APS (dangerous to touch, inhale or ingest) and water (wet, translucent) I was ready to assemble the array. After a complex procedure involving plastic locks, glass screens and electrodes, it was plugged in and Dr J and I injected samples and markers. Unlike in first year, nothing went horribly wrong and I managed to leave in the knowledge that the previous day's work had not been ruined by my previouly departed and unmourned tendency to inject into the buffer.
So once the gel was set, it was removed and sandwiched between a load of filter paper squares and sponges, inside some sort of holey thing. Refusing to be overcome by the technical nature of this task, Dr J and I ensured the nitrocellulose membrane the proteins in the gel were to transfer onto was facing the correct way, as filter paper is probably a worse adsorbant in terms of being able to wash it for several hours without it disintegrating. Then Dr J plugged in the electrodes, turned constant voltage up to 100V for an hour, and left it to run.
Now this may look mundane, but if only you knew what was going on inside that box. Sizzling hot electroblotting, baby.
There are two types of protein ladder markers, one are visible and coloured, whilst the other is detectable by antibodies and shows up with the rest of the protein you're trying to detect. The coloured markers transferred pretty much entirely, which Dr J said was a good indication of an upcoming good set of results. Although obviously there're other factors involved.
Afterwards we washed with a generic protein buffer to minimise any non-specific binding which would throw the results, a primary antibody which latched onto the tagged ZipA, and a secondary antibody which in layman's terms is shiny as hell. This secondary antibody is linked to horseradish peroxidase which interacts with hydrogen peroxide and a colourful acid to give off a signal detected by camera. But that's a story for tomorrow, as these washes took around an hour per cycle.
Meanwhile, my observation of lab hazards has been progressing. Whilst acetic acid (vingear) is to be found in various containers throughout the lab, the generic protein buffer was comprised of 1.25g of milk powder (semi skimmed) in 25g of PBS (1X). I don't like milk so I forsee no accidental consumption of lethally spiked tea. But what if you're lactose intolerant? Always wear gloves.
Thursday, 1 July 2010
My project this summer is on the divisome, which is the structure responsible for bacterial fission. Specifically, it's on FtsA and how it interacts with FtsZ.
Fts stands for Filamentous Temperature Sensitive, and describes how when heated these proteins form filaments! [EDIT: No they don't, see later to find out why!] More relevently, FtsZ polymerises to form a massive ring, which contracts via a GTP/GDP dependent mechanism, bringing the membrane with it, until it draws tight like the drawstring of a sleeping bag, pinching off two distinct bacteria. FtsA is a membrane-associated protein that mediates division by binding to FtsZ, but is not essential for this process.
So, technical sciency part over; what has my first day been like? Well after realising cheese and pitta bread sandwiches for breakfast was a mistake, I arrived at the lab where I met Dr. D and was filled in on the basics of what I'll be doing, at least for a few weeks. Then I was introduced to the post-grads who all seemed pretty nice people, as well as the technician who was given permission to shout at me. The ground rules for the lab are:
- No open-toed sandals in the lab
- No food in the lab
- No drinking the technician's coffee, ever
- Keep the lab tidy
- No lab coats in the research office
After smuggling my lab coat out of the research office I entered the lab, where Dr. Mohammed Jamshad (also a really nice guy, and my de facto mentor for this project) showed me around. He's currently harvesting ZipA proteins from E. coli, which are acquired in a similar manner to the FtsA I shall be harvesting later, as well as sharing similar roles (possibly).
So first we removed the growth media from the autoclave (a sort of pressure cooker used to sterilise things) and added ampicillin to kill any unwanted microorganisms that might sneak in when our backs were turned. To this we added 1% volume of a culture of E. coli, transformed with a plasmid containing ZipA and ampicillin resistance. Once this was done, we stuck them in an incubator and swirled them for about 3 hours at 37 degrees celcius and at 200rpm, securing the flasks with wads of tissue stuck between the vessel and the holding ring. That thing looks like it'd be unpleasant to clean, so I'm definitely going to remember to do that.
Then we cleaned up the glassware. In order to clean the culture flask Dr. J used a pink chemical which was presumably some sort of bleach, adding some to water and then swirling it in the flask, before leaving it for 30 minutes.
Now, it is here that I noticed a lab-based hazard. If there's anything I have learnt from organic chemistry practicals, it is that very dangerous chemicals tend to look highly edible (my substituted phenyls tend to look like gingerbread, although this is probably down to appalling technique). Moreover, laboratories have a tendency to make me very hungry (again, probably due to lab sessions traditionally ending at 12, lunchtime). Finally, this bleachy stuff looked and smelled of sherbert.
I voiced my concerns to Dr. J, who reassured me that it did indeed look and smell like sherbert, but was also very bleachy, thus I should refrain from sampling it. I was learning more and more about lab behaviour and scientific technique by the minute.
At the moment I am on a short break, in a while I shall be putting together some IPTG to induce translation of ZipA, as well as checking the flasks to make sure they're not on fire, and that the optical density is about 0.6-0.8 (which I thought was surprisingly high).
First day is going very well indeed. :D