Friday, 9 July 2010

Just keep spinning...

The last few days have been eventful, and I have even more pictures to demonstrate this. Essentially I've been doing what I've done previously, but with much less help from Dr J. Whilst he's still been around to handle the more technical aspects of the procedure - operating the camera, reminding me where things are, explaining why what I'm doing is wrong/pointless - I've been allowed to do most things without much supervision.

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:

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