I'm sure I should feel happier...

Well...I handed my dissertation in. I'm sort of waiting for a nice happy heartfelt rush of relief, or some kind of feeling. Something other than cold and tired would be nice.

It did look all professional though; bound up properly with some nice results, pretty pictures and surprisingly meaningful graphs. And I think I'll probably feel better tomorrow, when I hand my lab book back to my supervisor in the safe knowledge that she's continuing with the work we were doing, and may yet find something even more interesting. At the moment though I'm kind of oscillating between oh-my-ghod-so-much-revision and well-there-goes-10%.

So if you want some interesting science go here for snails that ride on other snails, and other easy-to-understand scientific awesomeness from Ed Yong.

And if you want some mind-knotting philosophy go here for a discussion of induction that I feel so proud to actually understand (eventually and after much discussion)

(and if you want general fun, go to xkcd because it's always good)

Translocon structure

Well, I'm still busy with revision but, miraculously, I seem to have almost achieved my crazy aim of writing all of first terms lectures notes in a week *dies theatrically*. I still don't actually know any of the stuff, but at least I now have notes to work from, and I understand it all, which is important.

Topic of the day today was protein targeting within cells, specifically targeting secreted proteins to the inside of the endoplasmic reticulum; a network of internal membranes which modifies secreted proteins and then exports them out of the cell.

Best story in all this is about the discovery of the structure of the translocon; the channel the protein goes through to get into the endoplasmic reticulum (through a membrane). When they first started looking at the structure of the translocon, they saw it was usually found in the form of four proteins very close together, so the first idea was that these formed a ring, with a nice wide hole in the centre for the protein to travel down. It made sense; but unfortunately it only made sense in that specific biological way where the idea is nice but it doesn't fit in with biology.

Because from the cells point of view (if it has one) a large channel like that is a very unhelpful thing to create. You can't regulate it; it needs a filter, or a cap, or some mechanism to prevent just anything going through. Also, a closer look at the translocon showed that it wasn't always found with four proteins close together; sometimes there were three, or two, which would make the channel even less specific or (in the case of only two) almost non-existent.

So the current model (with a lot more supporting evidence) is that there is a little channel down the middle of each individual protein, shaped a bit like an hourglass, which secreted proteins travel through to enter the endoplasmic reticulum. This model works better, especially as the middle bit of the hourglass can expand and change shape; allowing protein folding inside the channel. This also allows for proteins that want to stay in the membrane to be released, there's a little exit space near the middle of the hourglass (alpha helix two of Sec61 for those who are interested) that allows the protein to escape from the translocon into the membrane before it enters the endoplasmic reticulum.

But of course every new model leaves questions behind that were answered by the old model. The thing now, is nobody is quite sure why the proteins cluster together in groups of (mostly) four. If they have a channel through each protein, why not be all separate? Why form specifically numbered groups? It has been suggested that one protein is used for recognition while the other is actually used for the channel but it's all a bit uncertain at the moment.

*sigh* I miss lab work. I miss blogging about lab work. Revision is like forcing yourself to eat when you're already full, I want to get onto some new stuff.

That Time Of The Year

Well, I am back now in the land of fast Internet, and doing that thing that happens when exams loom which is try to remember how the hell information is supposed to get from large numbers of bits of paper into your head.

I'm currently revising transcription, which I first encountered in AS level (aged 16 for those not familiar with the English schooling system). I've been taught it almost every year since as well, and over the years the process seems to have become more complex and less certain (along with everything else, strangely enough).

Transcription is the first step for making proteins inside the cell. The information for creating proteins is stored in DNA (...mostly..more on that maybe later), with every three base-pairs of the DNA coding for one protein amino-acid. DNA is made of a string of base pairs held in place by a sugar-phosphate backbone, and proteins are made of strings of amino-acids all folded up so it works quite well.

However the cell doesn't just make protein from the DNA template, it goes through an intermediate step first, making an RNA template of the DNA (known as messenger RNA, mRNA). This is the process of transcription (link leads to a nice animation). The RNA then leaves the nucleus and is used for a template to make the protein.

One thing you get taught in AS levels is about promoters. Promoters are regions of DNA that specify the start sites of transcription, the place all the transcription machinery binds too, before trundling off along the gene. You get told that they have a things called a TATA box, ten base pairs away from the start; essentially a conserved sequence of bases that bind to the transcription machinery very well; and conserved (ish) bases around the start site called the INR box. This makes sense (especially when they tell you how the machinery actually works) and specifies exactly where the mRNA should start being made from. Here's a paper.

Except it turns out that these TATA box promoters are a really rare form of promoter. Most promoters are a lot less precise, very fuzzy, and the start site can be anywhere within about 20 base pairs. The mRNA that comes out frequently has extra bases at the front end, because the start point is not well defined.

This is mentioned very briefly, and then they tell you everything and more about TATA box promoters all over again. This is because people know about TATA box sites, because most if not all of the research is done on them, and that is because all of the focus is on them. Also getting ideas out of scientists is a lot, lot harder than getting them in, and the nice preciseness of the TATA box promoter is a lovely idea. It's just not the one the cell uses the most.

Hehe. Science is crazy fun sometimes. Good luck to everyone else out there hitting exams as well. :)

Slow internet should DIE

I'm overseas at the moment, which means that although I am surrounded by palm trees, the weather is warm and I am practically living in the swimming pool, unfortunately I am reduced to dial-up Internet.

Which is very...very...slow.

So there will probably be a short break from blogging, as I'll be using the computer infrequently (although I am determined to message a Certain Special Someone every single day, even if they don't message back :p )

On the plus side, the lack of Internet means that I'm doing about six times as much work as normal. Which hopefully (hopefully!) should help with exams next term.

On Conferencing

I spent most of last week at a conference. The Society of General Microbiology conference to be precise, up in Harrogate. It was my first conference, so I was pretty excited about it, and it certainly lived up to expectations. It was fun, exciting, and I learnt a whole lot of stuff. Some of it was even about science.

But the most amazing thing? Julian Davies was there. The same Julian Davies I spent the last blog post ranting about; with the papers about how sub-inhibitory antibiotics acted as signalling molecules. Best of all, I had a chance to talk to him as well. After the initial slight embarrassment of me being slightly uncertain of how to introduce myself (mostly I was desperately attempting not to gush as him in an 'I've-read-all-your-papers-and-omg-they're-amazing' way) I finally talked a little about my research.

He actually seemed interested! It turns out all his work has been on soil bacteria, so he was quite interested in my work, which was on an antibiotic-producing bacteria. We chatted for a bit, then someone else came up and I politely scarpered out the way feeling a little wobbly around the knees. Gushing mostly avoided. Serious scientific talk achieved :) It was a good feeling.

One thing I didn't realise about conferences was just how much the evenings play a part. During the day there are talks and lectures and poster displays and promotional stalls for various companies that sell scientific equipment. So there is nothing official planned for the evenings (I even brought some revision along for then. hah. Like that happened). But the evenings, it turns out, are all about going out with the people you've met at the conference; getting to know people, talking with them, sharing experiences and ideas and having the most amazing conversations about scientific things with people who pretty much think along the same lines as you. And nobody rolls their eyes and tries to change the subject. It was amazing.

So that was my first conference. Hopefully, it will be the first of many.

They are talking to each other!

In my various readings and travelings through the complex and confusing world that is science, I have a tendency to pick up 'pet theories'. Theories that I think are so wonderful and fantastic and explanatory, and that make a lot of things that I'm doing make sense. I've never made any up myself (technically they are 'pet hypothesis' actually thinking about it) but I do steal other peoples.

My latest little pet comes from a series of papers written by Julian Davis; which look at the effects of low antibiotic concentrations on bacteria. Because antibiotics do exist in the wild, just in far lower quantities than are used in hospitals. The big question for a while has been what do they do in the wild. The common idea was that they were used for defense purposes, but they're usually released at quite low concentrations, only a few of them actually lead to death, and even then under very specific conditions.

[as an aside, Alexander Flemming (of penicillin fame) was very lucky to get the visual effect that he did. If the room had been slightly colder, or warmer, it wouldn't have worked. Naturally produced antibiotics at naturally produced concentrations are pretty rubbish when it comes to killing things]

So Davis's idea, which is an AMAZING idea, is that the antibiotics are used as signalling molecules. They are quite small molecules, which can diffuse relatively easily into the bacteria, and once inside they can effect which proteins the bacteria express. Take a look at the picture to the right (taken from this paper):
The little disks contain antibiotics, at low concentrations (known as sub-inhibitory concentrations because they don't inhibit growth). The colourful image on the right shows different levels of reporter protein. The antibiotics are effecting the level of protein expressed. If they can do it for a reporter protein, they can do it for other proteins in the cell. Antibiotic signalling could be a way for bacteria to find out and communicate information about their immediate environment, both within and between species.

Which is why the last week, back when I was still doing my project, my supervisor pulled our crazy-result plates out of the incubator, shook her head turned to me and said "Look. They are talking to each other!"

This is all epigenetics by the way, rather than genetics. The antibiotics are effecting which proteins the gene expresses, and at what levels, rather than changing the genome of the bacteria.The bacteria I've been working on, for example, have about 20 'silent genes' which don't get expressed in lab conditions, maybe antibiotic signalling would turn some of them on?

It's a lovely idea, and it fits in so well with the results I've been getting. I will reverentially place it with the Histone Code Hypothesis and the Aquatic Ape Hypothesis, in the place in my head reserved for pet theories.

How bacteria make antibiotics

There are many different types of antibiotics bacteria can make, but my lab project (now finished, alas) was concentrating mostly on a type called polyketides. These are not just antibiotics, some polyketides can also be antifungals and anticancer agents too, so it's not surprising that quite a lot of work has been done characterising their formation.

Here is the molecular structure of erythromycin. Like many polyketides it is circular, which at first appears to be a bit of a headache to synthesise. The way it's put together, though, is actually very clever. The backbone of the circular section is made up first, using a system of modular enzymes that pass the growing chain along like a conveyor belt, adding new residues at each stage. Then the straight chain is curled up into a ring, and finally the two side residues (the ones on the bottom right of the chemical structure, that look a bit like squashed rectangles with dents in them) are stuck on.

So here is the picture that has appeared on every slide show in every lab meeting we've had this term, showing the formation of the straight-chain backbone before it gets curved into a circle:
Ignoring the little letters (which are just names of enzymes) it really does look a lot like a conveyor belt. At each stage the chain is lengthened, before finally being taken off and twisted around onto itself (to form a circular molecule called DEB). The modular nature of this system is fascinating to work with, but a real problem to sequence. DNA sequencing techniques work mainly by chopping the genome up, sequencing the bits, then trying to stick them back together and modular repeats tend to confuse them.

The last stage, going from DEB to erythromycin, is just a matter of decoration. Although the squashed-rectangle additions (glycosylases, added by glycosylation I believe) look complex, they are quite common molecules that get added onto things in the cell. Glycosylase residues and glycosylation enzymes are very common.
And that's how bacteria make polyketides :)

Scanner Woes

How many times have I heard it on deviantart.com? The continual cry of "Oh noes! The scanner ate my picture!" And now I'm making the same complaints about mine.

Actually, my plate pictures haven't been too bad. Although a couple of them are somewhat ... darker and fuzzier than they could be. As there are no labels for anyone to pinch my results with, I think I am justified on posting a few on here:

That one's alright. A bit dark though. At least the contrast can be seen. In case anyone was wondering, what you are seeing is bacteria streaked across the plate and left to grow (marked by the black line across the plate) and the another bacteria grown on top of it, which has subsequently been killed.

But as this may turn into a paper (oh please! *crosses fingers and hopes*) I can't be any more specific.



This one (on the right) hasn't really come out at all. meh. There's not much I can do except tell people what they should be seeing and hope it all works out alright. So what you should be seeing is a patch of dead overlay (the light bit) that follows the line of the mould (crosshatched black pen), but is not actually present under most of the mould.


I have a dissertation to write up based on these! *panic*

On the plus side, my scanner seems to have also created some results of its own. Take a look at this bioassay:

See the white lines around the circles on the first two rows? They were not at all obvious in the photo. Although when I squint at the photo now I can kind of see them.

Yes, that is my handwriting at the bottom of the photo. Which was taken by yours truly in the (very old) lightbox, narrowly avoiding getting an accidental blast of UV light as well (UV light is used to take pictures of gels, and nobody bothers to switch the switch back to 'white light' when they've finished; noticed just in time)

I am very proud of all my results :) Which has probably confirmed for a Certain Special Someone that they are indeed going out with a very nerdy little thing.

=D

Yes but what does it do...

I am currently trying to get myself to finished writing an essay (rather terrifyingly my first essay of term) on the different approaches to gene annotation in vertebrates. As I've just woken up (afternoon naps seem like such a good idea until you wake up with a mouth that feels like a hamster died in it) I thought I'd give a quick summary of gene annotation methods:

Gene annotation is the 'interesting' bit of genomics. Quite a lot of gene sequencing work has been done, some of it (especially the human bits) very highly publicised. And while genome sequencing is probably useful (more on that maybe in a more ethically-inclined post) on it's own it's not terribly exciting. You're left with a big database full of mindless streams of nucleotides and one bit embarrassing question:

What does it all do?

Gene annotation attempts to answer that; trying to work out which proteins each gene codes for, essentially what the end function of the genome is, what each piece of DNA is used for. There are two main methods: just using DNA, and using data from protein/cDNA sources. Both of these methods can be either comparative or non-comparative:

1) Just using DNA: Non-Comparative
This relies on getting a program such as GENSCAN to, quite literally, scan along the DNA looking for the beginning and end of genes based on sequence patterns it had been told to recognise. Not so good for function, but useful enough for finding the damn genes in the first place. Also relatively cheap and you can go run it overnight.

2)Just using DNA: Comparative
Like it says, this compares your DNA with other previously annotated pieces of DNA to see if there are any very similar bits it can ascribe function to. It's a good starting point, especially now the pool of annotated genomes is increasing, but it's really bad at finding gene start point, especially when there are 'introns', or bits of DNA that are not actually turned into protein. Which is around 95% of the human genome incidentally. (an e.g of this, if anyones interested, is TWINSCAN)

3) cDNA/Protein data: Non-comparitive
cDNA, just to clarify, is DNA that has been reverse transcribed from RNA templates; i.e itt's all the DNA that will get turned into protein, and without any of the introns. A good way to use this is to make cDNA 'libraries' i.e all the cDNA within the cell stored on plasmids, choose one at random, see what it makes and, at the same time, find where it is in the genome. Simple and useful.

4) cDNA/Protein data: Comparative
This compares your genome with bits of cDNA from other genomes, where the cDNA has known function. Protein comparison is even more useful as seeing what protein your protein most resembles provides structural information, as well as functional and allows you to build up homologous families of proteins with similar function (if you have enough genomes). Also if you have enough protein data you can say you're doing 'proteomics' and the more 'omics' words in your project, the more funding you're likely to get :)

By the way, all of these comparative methods are based on homologous evolutionary relationships between the genomes, so anyone who says that scientists never use evolution is WRONG. (and probably pissing off the evodevo people as well)

As always, any questions are welcomed, leave them in the comments and I'll get back to you.

Disclaimer: This post was written while half asleep. Any spelling/grammer mistakes are therefore completely the fault of the writers Brain On Sleep.

Jumping DNA

Here's a bit of random information while I wait for my agar to melt...

Bacteria have a remarkable genome plasticity. They are able to mop up spare DNA in the environment, take pieces from circling bacteriophages (viruses that infect bacteria for the uninitiated) and exchange parts of their genome with bacteria from another species. The species boundary can be very wide as well, the bacterial equivalent of a mouse nicking bits of DNA from an elephant and incorporating it into it's genome.

In order to jump from one genome to the other (bacteriophage and other viruses) the DNA must be flanked by so called 'transposable elements' usually shortened to 'transposons' because molecular biologists are lazy when it comes to saying unnecessary words. (also, I suspect because 'transposons' sounds more scifi and scientists have a distressing tendency to be geeky like that). These transposons code for enzymes that cut the DNA out and paste it elsewhere, essentially allowing it to jump around between various genomes, being expressed and replicated in different bacteria.

ooop, there goes the sodding microwave. We have a new one after the old one stopped working (about three weeks after I entered the lab. PURE COINCIDENCE) and this one hits a pitch which is just slightly higher than the comfort level.

Incidentally, does anyone know how to do those fancy 'cut' things in blogs? Like when there's a blue underlined 'read more' label which whisks you away to the rest of the blog post. I'd really like to do that but I don't know how...