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...

Antibiotic resistance

So, in the interest of actually doing something about the John Rose Essay (as I haven't done anything yet). I thought I'd take a quick look to try and determine what information was generally out there about antibiotics. First stop wikipedia:

• 1 Causes and risk factors
• 2 Mechanisms
• 3 Resistant pathogens
  • 3.1 Pseudomonas
• 4 Role of animals
• 5 Alternatives
  • 5.1 Prevention
  • 5.2 Phage therapy
• 6 Development of new drugs
• 7 Applications

It's not bad. They spend a lot less time on the mechanisms than I'd like too, and far more time listing the main Bad Guys of the resistance world. There's also a lot more they could put under 'applications', but I can't write too much about that because that's what I'm researching at the moment :)

Next stop, the website of the department of health. They are currently running a new 'awareness campaign' (who knew that?) to try and discourage people from using antibiotics when they don't need to, i.e for colds and things. They had a previous campaign featuring a little talking drug which unsurprisingly seems to have petered out. You can download the leaflet here, but I wouldn't get too excited. It very closely resembles old WW2 posters, the kind telling you to eat more carrots or put up blackout curtains.

The current campaign seems to involve brightly coloured posters with simple messages on the front such as "Unfortunately, no amount of antibiotics will get rid of your cold". Less...snappy than a little talking drug shouting 'don't wear me out!' but a lot more adult somehow.

The Health Protection Agency's website I am a lot less impressed with. It's very...orange. And doesn't actually have very much written about what is quite an important topic. It does have a 'recent updates' thingy but this is pretty much the entirety of what it says about antibiotic resistance:

"Antimicrobial resistance describes the ability of a micro-organism to resist the action of antimicrobial drugs. This is important as it can make the treatment of infections more difficult and increase hospital costs. Undertaking laboratory testing of organisms causing infections can play a role in deciding the most effective treatment options."

wtf is with that last sentence?

So, having taken a brief look at the competition (I have to go pour 72 plates now!) I think I'll concentrate mainly on mechanisms with my essay. How antibiotic resistance arises, with probably a brief once-over of why somewhere near the conclusion.

hehe I have an Epic experiment planned. Hence the 72 plates.

Random thoughts

When I was young I used to keep a diary. Or a journal, whatever. I used to write in it most nights, usually about twice a week. I think I kept it up for a while, with some sort of wistful idea that spacemen, or future generations of humans, would some day find it and use it for scientific purposes, and in doing so make my life slightly more useful.

And in the manner of most people who write journals when they're younger I took a look at them over the holiday last winter and collapsed into a fit of laughter and the rather embarrassing hope that aliens or future generations wouldn't find it, or if they did would decently burnt it very quickly. I read through, fascinated at the fact that I'd spent so many evenings carefully and methodically writing down the exact same thoughts night after night.

Each day went something like this:

• Went to school today
• Came back and did some work
• I'm not working hard enough!
• I need good results!
• I don't really know if anyone likes me
• An amazing new book/film/lord-of-the-rings-associated-product just came out!
• I have so many story ideas in my head and no time to write them all...
  

Most of it was the fairly standard teenage mindset preoccupied with death, sex and the occasional foray into religion. At one point I even made up my own religion because I wanted to Believe in something but didn't like any of the mainstream ones. And inordinate amount of time was spent worrying about biology IGCSE's and (yes I did keep it up that long) A-levels.

All fairly standard, all fairly normal. Only one thing, in fact, was scary.

These are still the same sort of thoughts I'm thinking now.

Although with slightly less emphasis on Lord of the Rings.

It's scary and it's spooky just how little my internal monologue seems to have changed. There's less of the death and sex, true, and slightly more of the I'm-not-working-hard-enough but overall my deep internal monologue seems to have survived the experience of growing-up relatively unscathed. Deep down, I am the same sad nerdy little person I always was :(

On the plus side though I am working with tiny little plates! Petri-dishes five centimeters across. They look very small especially next to the giant petri-dishes which are 14 centimeters diameter and therefore awesome.

Lab Rat Writing

Having missed a couple of the other deadlines for Science Writing Prizes, I am determined to manage to get something to submit for the John Rose Prize which (in it's own words) gives an award for:

"The best explanation of a scientific principle of general interest"

Anyone have any ideas about what I should write about? At the moment, I'm leaning towards antibiotic resistance because a) it's in my course and b) it's useful.

Or maybe I could write about Biorefineries, and just reuse bits of my essay? Phage therapy? Although that isn't exactly general interest at the moment unfortunately.

At the very least, writing this in my blog should give me enough commitment too it to actually write the damn thing instead of just waving at the deadline as it passes.

On feeling ill...

I suspect having a cold isn't fun whatever job you're in. Coughing and sneezing in public tend to make people look at you oddly, and try to discreetly shuffle away in case they catch it. In a lab however, it's a whole new level of awkward because everyone around you is working with little eppindorfs and petri-dishes and trying their hardest not to get them contaminated.

Contamination is the One Big Evil in microbial biochemistry. And I've essentially turned into a walking contamination spray. I am already worrying for the fate of the plates I poured today, and it's only a matter of time before people start following me around with ethanol'd paper towels and disinfect everything I've touched.

I'm even starting to want to do it. Thankfully most of my work is done under a laminar hood, which is like a large box with a glass pane in the front which separates me from the plates and the bacteria. Which means that if I cough at the ceiling my plates should manage alright.

If they get contaminated I will scream. This is already the third time I've done this procedure. The first time the actual antibiotic we were using was contaminated, so we just grew nice little colonies of black mould. The second time I forgot to dilute the mixture down, so I was pipetting tiny, tiny amounts of liquid out and all the results were screwy. The problem is, is that the bacteria take five days to grow, which means I've already wasted two weeks and have no results.

And I'm ill. Meh. Welcome to science, two steps forward, six steps backward. :(

Where does the disk go?

Part of what my supervisor is doing for her project involves growing bacteria then challenging them by putting little antibiotic disks on the plates and seeing if the bacteria die or not. She can't make it in today, so she asked me yesterday if I could put the disks on the plates for her.

The procedure was fairly simple and wouldn't take long, so I agreed. I came in this morning all bright and early, and suddenly realised I had no idea where on the plates I was supposed to put the disks.

It the middle? At the edge? Where the bacteria are growing?

It's my fault for acting so competent in my first week. It made her form the impression that I know what I'm doing. :(