Lab etiquette and priorities...

I've been blogging quite a lot about actual science lately (which is good) so I think I'm excused one quick blog post about being a scientist, or in my case, being a Lab Rat. The thing is that I don't rank particularly highly in the lab; I'm doing a one year project, with not a huge amount of significance and in terms of funding I'm pretty sure the ants underneath the floorboards get paid more to be here than I do. And while I personally like to think I'm doing something useful for the lab I'm in, they'd probably be reasonably grateful if I served my time without causing any major damage, or using up too many resources.

So what do I do when (as happened just now) someone asks me if they can use the hood for the afternoon? And in a way that suggests that they kind of really need to use the hood that afternoon...

(Hood = laminar flow hood, which is a sterile working space).

What I did was to internally rearrange my schedule inside my head, to free up space for them. But what if that hadn't been an option? What if I'd been doing an infinitely-long colony pick or something? Who, in that situation, has the priority?

The PostDoc with their relevant research, or the Lab Rat who, by an accident of bureaucratic shuffling, happens to actually be in the lab that owns the hood? (there's about three labs in the space upstairs and we tend to share equipment)

I quickly finished up everything I needed to do right then, and then told the aforementioned PostDoc that she could use the hood whenever. Except...I still have more work to do in there this evening. And she hasn't actually started work in there yet. I don't want to start my work in case she suddenly appears, but I have no idea how long it is polite to wait before getting naggy at someone. Especially when that someone is a) older than me b) doing more important work for the lab and c) bringing more money in for the lab.

I have decided to deal with this problem in my usual way of dealing with all problems, by scuttling away and hoping it disappears in it's own time if I stop looking at it. Which is why I'm currently sitting downstairs in the dry lab using the computer and consoling myself with vending machine chocolate bars. I can't really go up until either she's finished with the hood or my E. coli plates dry, and they won't for a while because I accidentally massively over-poured one of them.

On the plus side, I discovered why my transformations didn't work yesterday. I plated them out on the wrong antibiotics and they all died. In my current state of mind, I can't help but find this kindof hilarious.

Amphibian Skin

I decided to take a break from bacteria today and decided it might be fun to just choose something totally random to write about. Taking a daring leap into the unknown I decided not only to try and find out somthing about multicellular creatures, but about those multicellular creatures about which I know the least: amphibians.

The above picture of an axelotl may have had something to do with my decision. Note the similarity to a pokemon that has accidently wandered into Star Wars.

There are three main orders of amphibians; salamanders and newts, toads and frogs, and caecilians; the blind legless ones that live at the bottom of caves. They are cold blooded and, unlike many other multicellulared animals, they don't regard the outside environment as completely seperated from the inner. They can exchange both water and oxygen through their skin, in fact some salamanders exchange all of their oxygen in this way, and thus don't have any gils or lungs at all.

Skin is therefore a very exceptional organ in salamanders, as it is used for fluid balance, respiration and the transport of essential ions, as well as the more traditional uses of protection and sensing. Possibly because of this, it has it's own protection system against infection. Amphibians have both an adaptive and innate immune system, but in addition to this they have granular glands under the dermis layer of the skin that release antimicrobial peptides in response to stress. Peptide release is stimulated by the adrenergic receptors, so any circumstance of shock of pressure results in an extra layer of protective peptides over the skin surface. They are quite potent as well, providing potential protection from bacteria, fungi, protazoa and even viruses.

As cold-blooded creatures have a slower reacting immune system, this quick, automatic and generic response to stressful conditions provides important protection for the skin, which is vital for maintaining internal homeostasis. And as well as peptide-releasng glands, they also have pigment granules under the skin, which give them bright colours and mean they can change between colours depending on environmental conditions or what they want to communicate.

Amphibians have such beautiful colours...why do they always make dinosaurs blotchy khaki!

There is however a downside to using your skin to take things up from the environment. Water isn't the only thing that gets through permeable skin, chemicals dissolved in the water can as well, which can be fatal if the chemical in question is a herbicide such as atrazine or glyphosate. Chemical contamination may be one reason (and there are, no doubt, many others) for the dramatic decline in the number of amphibians over recent years. Apparently conservationists are majorly concerned about this.

And they don't seem to get as much press as endangered mammals either. Which is a pity because that axelotl does look quite sweet. And amphibians are the only living proof we have left to remind us that the dinosaurs could have had bright-coloured polkadots:

Imagine those colours....on a velociraptor!

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Rollins-Smith, L. (2005). Antimicrobial Peptide Defenses in Amphibian Skin Integrative and Comparative Biology, 45 (1), 137-142 DOI: 10.1093/icb/45.1.137

Quaranta A, Bellantuono V, Cassano G, & Lippe C (2009). Why amphibians are more sensitive than mammals to xenobiotics. PloS one, 4 (11) PMID: 19888346

Idea Space

One of the things about working in a research lab, is that it's very easy to get lost in the little world that you're currently working in. Day to day research really is little-picture stuff; while you know that your work has bigger implications, and you drag them out the filing cabinet every time you want to write a grant application, you don't often have the time, or the inclination, to just sit down and think about where your work could go.

This is why scientists need friends. And I discovered over the summer that this especially applies to friends who are also art and design students. Because while you're busy squinting at gels and trying to convince yourself you have a band 2kb long they are getting excited at the fact that you have purple bacteria, actual purple bacteria, and they're thinking of all the amazing things you can do with that.

I'm still recovering from jet lag a little, so I'll try to put this in context just using pictures. This is what I see:
And this is what they see...

Scatalog picture from the E.chromi design team, more details here

Once you've stopped sniggering at the fact that it is a case of coloured poo, it starts to dawn that this is actually a very elegant system for searching for intestinal problems. taking bacteria that turn different colours in response to different conditions can result in a full spectrum (as it were) of the conditions in your stomach, just from looking at your poo. And doctors generally do look at poo to check how a patient is doing, this just gives a clearer picture.

And once you start thinking about it, there are a huge number of applications for coloured bacteria. Here's a few I've thought up over the course of the last few weeks:

• Putting the colours into spores (e.g from B. subtilis) could give you little dots of colour: bacterial pixels
  
• Industrial fermentors use bacteria and yeast. Adding conditional-dependant colours could allow you to check the conditions (i.e temperature, pH) without needing monitoring equipment, the bacteria just tell you themselves.
• Bacterial pigments for the pigment industry in general, there is a whole range of different colours in nature, you could make them into paint/dyes/etc just with a fermentor.
• Moving bacterial art. Bacteria swarm in the direction of food sources. Swarming coloured bacteria would be awesome, they'd look like that bit near the end of 2001:A Space Odyssey where he does the trippy planet landing.
• And of course, environment monitoring. Get lead sensing bacteria, drop possibly-contaminated water on them, leave in the incubator overnight and if the thing turns bright red the water isn't safe. Easy, convenient, and potentially quite cheep.

There's probably many more, whole worlds of idea-space for potential applications. They're just a little hard to see when you're standing two inches away from a gel, squinting through an ethidium bromide visor.

This is a massive shoutout to the two design students (who know who they are!) who helped me and my fellow lab rats retain our sanity over the holidays. You took our humble experiments and took them in such wonderful, marvelous directions, and at the end of it all you managed to get a case of coloured poo past Heathrow Airport security. And to all design students anywhere who are working with science; trust me, we need you. :)

America!

I was hoping to get another paper-analysis post in before I left, but I ran out of time. I'm off to America tomorrow morning (*early* tomorrow morning) for a synthetic biology conference. I get the feeling it's going to be utterly mad, and leave me completely exhausted by the time I get back (on Tuesday evening).

Expect some residual synthetic biology stuff when I get back! I'm hoping to scribble down enough for a post while I'm there, and type it up when I get back. I could bring my laptop along, but I'm trying to keep my luggage down to hand-luggage and I suspect I wouldn't have the time. Also, I'm not quite sure of the etiquette of conference-blogging. Some of this stuff might have publishing-potential but not yet been constructed into a paper, and I don't want to accidentally 'out' someones research.

As a quick teaser, here's a picture of what me and my fellow summer-project lab rats will be taking about. All the pigments were made in E. coli:

NextGen sequencing: What Is It Good For?

Sequencing DNA has become a major industry. The genetic code of an organism contains huge amounts of data, and the potential for a greater understanding of how it works at an intracellular level, and whole centers and genome sequencing factories now exist to fill this need. While most of the sequencing is still done using a modified and more efficient version of Sanger's original dideoxy method, next-generation sequencing machines are starting to emerge that can achieve what is imaginatively named massively parallel sequencing. Massive amounts of DNA can be sequenced in parallel, and we're talking MASSIVE amounts of DNA. Illumina/Solexa machines can sequence hundreds of thousands of DNA molecules all in parallel.

The basic Sanger sequencing method is shown below (image taken from the Science Creative Quarterly, which also has a very good description of the process for those more interested in DNA sequencing)
There is a catch in massively parallel sequencing however. Sequencing works by breaking a large DNA molecule down into smaller 'reads'. Each read is then sequenced and they can be stuck back into the right order (with varying accuracy) once all the reads have been completed. Sanger sequencing (diagram above) can produce reads up to 1000 base pairs long. NextGen sequencing is lucky if it manages 350 base pairs. They tend not to be quite as accurate as well.

What they are is cheap. Which gives geneticists an important tool; large numbers of short genome reads generated at very low cost. While these NextGen techniques are being improved, and there are many people looking into making them more effective for de novo gene sequencing, they are also being put to use in other areas, where the ability to sequence large numbers of short genomic sequences at low cost is hugely beneficial.

The most obvious areas are those where you don't need a particularly long sequence, such as when you just need to find the site of origin of a particular length of DNA. This is particularly useful for looking at transcribed portions of the DNA (those parts that are actually turned into proteins). Sequencing short bits of the transcribed RNA copy (that is used to make the protein) allows this to be compared to the original DNA sequence to find where the DNA corresponding to the protein is and, possibly more importantly, concrete evidence that it is being transcribed. In this situation the short reads aren't a problem, although there are still issues with the accuracy.

Another application is to look for novel small RNAs. These are small sections of RNA which regulate gene expression. They are discovered fairly recently (in plants originally) so there's quite a lot of excitement about them. As they're only small the length of the reads are not a problem. Pyrosequencing (a form of NextGen sequencing) was used to discover the Piwi-interacting RNAs, which are linked to transcriptional silencing in germ line cells.

NextGen sequencing also has a role in protein coding gene annotation. Protein-coding genes can be quite long, and would require several reads from NextGen techniques, but the low cost of these methods means that they are starting to be used for annotating protein coding regions. Integrating them with paired-end sequencing (which allows the reads to be re-connected more easily) removes some of the problems are shorter reads, and novel techniques are continually being explored to increase the accuracy.

NextGen machines are also starting to be used more for metagenomics, which works by taking random soil or water samples and sequencing every bit of DNA you can find, regardless of which organism it comes from. A metagenomics project in the Sargasso Sea (strangely enough most of these projects tend to take place in warmer climates...noone appears to do metagenomics in, say, iceland) produced over 1.2 million unknown gene sequences. These are suspected to be from 'unculturable' bacteria, which for some reason just don't grow in the lab, and metagenomics has revealed a huge number of these bacteria within the ecosystem.

If you want a novel genome sequenced your best bet is still to send it down to the Sanger Centre and be very polite to everyone who works there, but the growth of cheaper machines with massively parallel sequencing provides a whole range of new applications. Even if NextGen machines never quite reach the accuracy and read length of Sanger machines, there are still many areas in science to which they provide a large benefit.

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EDIT: I have been informed by people who know a lot more about this than me that NextGen sequences are now pretty much exclusively used for whole gene sequencing. It appears my knowledge is a little out of date. However this post is still an interesting exploration of the other applications of NextGen sequencers, so I'll leave it as it stands.

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MOROZOVA, O., & MARRA, M. (2008). Applications of next-generation sequencing technologies in functional genomics Genomics, 92 (5), 255-264 DOI: 10.1016/j.ygeno.2008.07.001

Hutchison, C. (2007). DNA sequencing: bench to bedside and beyond Nucleic Acids Research, 35 (18), 6227-6237 DOI: 10.1093/nar/gkm688

Making Mutants

Nowadays there are many different techniques for looking at gene and protein structure and functions. You can make protein crystal structures, you can see what substrates the protein binds too, you can do various chemical assays to open the protein up and see what it looks like inside. The most classic way however is the scientific equivalent of hitting it until it stops working, and then seeing what you've damaged. This technique, in a slightly more sophisticated wording, seems to be the cornerstone of much of biochemistry, and probably developmental biology as well.

In most cases the 'hitting' is a lot less random than I've probably made it sound. Say you have a stretch of DNA that binds to a protein, and you want to know which parts of the DNA actually physically bind to the protein. The best way to do this is to get the sequence of interest and change the base pairs (that make up the DNA sequence) very specifically, to see which changes stop the protein binding.

Starting with the hypothetical DNA sequence AATATAT. In order to find which bases bind to your protein, you need to make a few very specific point mutations. The way most of the labs around me do this is with a kit from Stratagene called QuikChange (R). Your DNA sequence is likely to be inside a plasmid (small circular piece of DNA) so you design a small primer with the change you want to make, i.e AAGATAT. Adding this to the plasmid, along with some polymerase to expand the DNA, and some nucleotides to expand it with and you get a perfect copy of the plasmid, perfect except for the small difference of the T-G mutation.

QuikChange provides plasmids for you to put your DNA sequence in, and these plasmids have been methylated; some of the DNA base-pairs will have methyl groups attached. To get rid of this original plasmid (after all, you only want your mutated copy, not the un-mutated original) you use a restriction enzyme (DpnI) that literally chops up methylated DNA, leaving nothing behind but your mutated sequence.

Then you add your protein, and see how it binds. If the binding is still just as strong, then that clearly wasn't an important residue. If the binding is weaker, or if less of the protein binds, then that might have been one of the important ones.

(You can of course just use PCR with mutated primers to create single mutants. But you do run a risk of introducing other accidental mutations through the PCR process. And when it doesn't work it's incredibly irritating. I did some research over the summer which proved conclusively that it is possible for PCR mutagenesis to not work for a continuous period of over two months)

Protists and their plastids

A quick skim through this blog reveals fairly quickly that I have a slight fixation on bacteria. I like to research them, read about them, and then blog about them, most specifically about their cell walls. However life contains more than just bacteria, and occasionally, strange though it might seem, people write papers about such non-bacterial things, and they end up on my desk with a small post-it attached reminding me that I have a presentation for my supervision group coming up.

So for the sake of my supervision, and to prevent myself becoming too scientifically blinkered, I took a quick foray this weekend into the murky world of protists, the strange and wonderful organisms that occupy the taxonomic equivalent of the 'misc.' draw in a filing cabinet. The creatures that are neither plant, nor animal, nor demonstrably bacteria. Many of them are single celled, some of them photosynthesise, and they all seem to occupy little evolved niches of their own, producing proteins with no noticeable homologues in any other branch of life.

The paper has the rather terrifying title of : "Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium". And I am not ashamed to admit that I had to go double-check the meaning of several of those words.

Dinoflagellates are little organisms that live in water, and mostly look a little like the picture on the right. Many of them are marine organisms, making up a large amount of the photosynthesising biomass in the ocean, and occasionally blooming to form 'red tides', leading to whole sweeps of water turning bright red (possibly occasionally on biblical command). The photosynthetic ones contain chloroplasts, which are wrapped up in three membranes, rather than the usual two. These, like all chloroplasts, contain their own genetic material (known as plastid genes), although unlike plant plastids, they don't seem to contain very many, and those that they do posess are found on little minicircles.

What the paper is interested in is whether there are any other genes in the chloroplast which aren't in minicircle form. There are, afterall, only 12 genes encoded on the minicircles, which is a small amount for a plastid. In order to explore this, it uses a characteristic property of the dinflagellate species it's working with. All organisms, when making proteins, make them from an mRNA copy of the genetic code. This mRNA copy tends to have a long string of adenosine residues added to the end, in order to prevent the mRNA getting degraded. This happens in our dinoflagellate species as well, but it doesn't happen to the plastid genes.

However instead of getting multiple adenosine repeats the plastid genes get multiple uracil repeats. It's just a different base, but it allows the mRNA made in the nucleus, and the mRNA made by the chloroplast to be separated. You can probe for adenosine enriched and adenosine depleted mRNA as shown on the gel below (A and B show different species). The psbA mRNA is clearly strongly present A+ (adenosine enriched) and therefore codes for a nuclear encoded protein. Conversely, the 23S RNA is A- (adenosine depleted) and is coded for in the chloroplast, from a plastid gene.

(Image taken from reference below)

The paper selected 300 random poly-uridine mRNAs (A-) and sequenced them to see if they corresponded to genes found in minicircles, or whether they might be plastid genes held in some different architecture. All the A- mRNA corresponded to the 12 genes discovered in the minicircle. They carried out rarefaction analysis to see if their sample size was large enough, apparently it was, in fact 300 clones was way in excess of the amount needed to find a further, non-minicircled-gene.

This suggests that minicircles are the only architecture for plastid genes and, importantly, that there really are only 12 genes contained in the chloroplast of the dinoflagellate Lingulodinium. This is a very small number of genes, all the rest have somehow migrated to the nucleus, leaving these 12 behind. And it's still very much an open question about why these have been left behind. The paper, in its discussion section puts forward the possibility of size. The genes that have been left behind all code for some of the longer proteins usually found in chloroplasts, although the paper does have the good grace to admit that that's not the most convincing of arguments.

It's worlds away from my little bacteria. But still just as fascinating.
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Wang, Y. (2006). Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium Nucleic Acids Research, 34 (2), 613-619 DOI: 10.1093/nar/gkj438

Damage Response Systems

Antibiotics can attack many targets in bacteria, and one very popular targets is the bacterial cell wall. Bacteria have been fighting natural antibiotics (produced by fungi and other bacteria) for millions of years, and have a variety of genetic strategies to aid resistance against synthetically developed drugs. Cell-wall antibiotic defence strategies fall into two major responses, which I'll illustrate with the example of bacitracin, as this is the antibiotic I've been studying for my lab work.

Firstly, a specific response against the attacking antibiotic. These can take the form of antibiotic degrading-enzymes, or efflux pumps, which move the antibiotic out of the cell. In the case of bacitracin, it's an efflux pump (encoded by the bcrABC cassette), which uses energy from ATP to transport the bacitracin out of the cell.

The most interesting thing about this system, and indeed many of the antibiotic-specific response systems, lies in it's evolutionary origins. The cassette originally came from a bacteria called Bacillus licheriformis, which is the bacteria that makes the bacitracin antibiotic in the first place. Soil bacteria tend to make a huge number of antibiotics, for defense and invasion, and if you make an antibiotic, it's a good idea to have some way of ensuring that it doesn't destroy your own cellular systems. In fact, given that this is an efflux pump, it might not even have evolved as a defense mechanism...just a pathway for moving the bacitracin into the environment once it had been made, as it is a secreted antibiotic

These ABC transporter systems are found fairly frequently as well. In B. subtilis (one of the better studied bacillus bacteria) eight out of the forty antibiotic genes have ABC transporter systems next to them. Because unlike in eukaryotes (like people) who can often have genes for similar systems on wildly different parts of the chromosome, bacteria like to keep genes used for the similar functions close together. They don't have much genome, they don't have the space pr the protection of a nuclear cell membrane, so they have to be more efficient about packaging.

The second type of response is a more generalised system; rather than responding to a particular antibiotic, it is instead a cellular response to the damaged cell wall. As an example the LiaRS system (a two-component response system)is activated in response to four different cell wall attacking antibiotics (all of which interfere with the rate limiting step of cell-wall building, the lipid II cycle). The Sensor (LiaS) has a short histadine kinase domain which is buried in the membrane. This recognises membrane damage and uses the energy from ATP to phosphorylate the Response Regulator (LiaR) which then leads to gene activation.

The Lia system is more than just a two component system however, there is a third component. As well as the sensor and responder, there is a third protein LiaF which keeps the system 'switched off' when the cell wall is not damaged. This is shown diagrammatically below:

Image from second reference (Jordan et al 2006)

When the cell wall is damaged, the LiaF inhibition is removed, and the LiaS can phosphorylate the LiaR, leading to a change in gene expression, which produces the appropriate response.

Unlike the specific responses, these pathways are often present within the bacteria, as a natural response to cell wall damage. These are not so much resistance mechanisms, as survival mechanisms, that are strongly selected for in times of antibiotic stress. The damage caused by clinical concentrations of antibiotic is usually too much for such systems to cope with, but they form an adequate defense against antibiotic levels in the soil.

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Ohki, R., Tateno, K., Okada, Y., Okajima, H., Asai, K., Sadaie, Y., Murata, M., & Aiso, T. (2003). A Bacitracin-Resistant Bacillus subtilis Gene Encodes a Homologue of the Membrane-Spanning Subunit of the Bacillus licheniformis ABC Transporter Journal of Bacteriology, 185 (1), 51-59 DOI: 10.1128/JB.185.1.51-59.2003

Jordan, S., Junker, A., Helmann, J., & Mascher, T. (2006). Regulation of LiaRS-Dependent Gene Expression in Bacillus subtilis: Identification of Inhibitor Proteins, Regulator Binding Sites, and Target Genes of a Conserved Cell Envelope Stress-Sensing Two-Component System Journal of Bacteriology, 188 (14), 5153-5166 DOI: 10.1128/JB.00310-06

How to destroy a bacterial cell wall...

The cell wall is extremely important for bacteria, as it allows them to maintain their existence as a single celled organism, and protects them from the harsh conditions of the outside world. Most bacteria cannot survive without a cell wall; which is why it's such a great target for antibiotics. And as the cell wall is constantly being recycled, disrupting the process to create new cell wall is as good as destroying whats already there.

Bacterial cell walls are made of strands of glycopeptide (shown below, picture from Kimball's biology pages) crosslinked together to form a mesh, which provides a strong support around the cell. There's a whole pathway of enzymes involved in creating the structure, and blocking them, or preventing them from working efficiently, is a quick and easy way of killing off bacteria.
This is the strategy used by Methicillin, an antibiotic that used to be talked about a lot a while ago (it's the 'M' in MRSA) but has been neglected by the media lately in favour of swine-flu and other viruses. Methicillin is a B-lactam antibiotic, which means that it binds to one of the enzymes involved in cell wall metabolism, blocking its active site. More specifically, it binds to the enzyme that creates the cross-links between the glycopeptides (PBP2). No new cell wall can be created, and therefore no more bacteria.

Resistance to this takes several forms. MRSA simply uses a variant of the enzyme, with a deeper active site, so that while the cell-wall precursor substrate can bind, the antibiotic cannot. Protection from a wide variety of different B-lactams can be achieved by B-lactamases, bacterial enzymes which break down the antibiotics. Multi-efflux pumps also exist, these are proteins that span the bacterial cell wall and essentially pump out any antibiotics that make their way into the cell before they can cause any harm.

Vancomycin is the drug that is still most commonly used against MRSA, although some resistance is (as always) beginning to arise. Unlike methicillin, vancomycin does not bind to any bacterial enzymes, instead it binds directly to the cell wall precursors. The part it binds to is shown below, as a close up from the earlier diagram of the cell wall:The incredibly inexpertly added D-ala in red at the bottom shows the precursor form of this section of the cell wall (Ala, Glu and Lys are the short-hand form of amino-acids, so this is just a short protein chain. The L- D- labels show what form the amino-acids are in). The vancomycin binds to the final D-ala-D-ala, preventing it from being processed and halting construction of the cell wall.

Two different methods of preventing cell wall growth; one antibiotic binding to the enzyme, the other to the substrate. And both, sadly, have been defeated by resistance already. In the case of the B-lactams, a wide variety of resistance mechanisms exist, from actively destroying the antibiotic, to pumping it out the cell, or bypassing the enzyme completely. In the case of vancomycin, some bacteria have started producing peptide chains ending in D-ala-D-ser, or D-ala-D-lac, and there have been reports of VRSA from America.

The pathway for creating bacterial cell walls contains multiple steps, and both methicillin and vancomycin halt just one of these, the cross-linking of the short peptide chains near the end. Bacitracin, another cell-wall directed antibiotic, works further upstream in the pathway. I've just been given a whole stack of papers on bacitracin by my supervisor...so I'll probably be writing more about that in the future!

Bacteria that use antibiotics...for food!

Antibiotic resistance is by now a well-known phenomenon. Resistance is carried in both antibiotic producing bacteria to protect themselves from their own weaponry, and the soil bacteria they attack, in an attempt to defend themselves. The sudden influx of pharmaceutical antibiotics has encouraged the spread of resistance to human pathogenic strains, leading to the so-called 'superbugs' seen in the media such as MRSA and vancomycin-resistant C. difficile.

However researchers at Harvard found that not only are some bacteria able to neutralise the threat of antibiotic resistance, they actually use antibiotics as a food source. Not only that, but they were capable of using antibiotics as the sole carbon source. The table below (taken from the reference at the end of the post) shows the survival of bacteria on antibiotics using samples from three different types of soil, Farmland (F), Urban (U) and Pristine (P - soil from non urban areas with minimal human contact for 100 years):

The antibiotics used include natural, synthetic and semi-synthetic molecules, all all of which could be used by bacterial species as a carbon source. Even more interestingly (or alarmingly) the antibiotics were at concentrations of 1g/litre, 50 times higher than the concentration normally used to test for resistance.

The 'pristine' soil is the one that the researchers found the most interesting, as the general expectation was that this area would contain fewer antibiotic-eating bacteria, having had minimal interaction with people and pharmaceutical antibiotics. However the data showed no noticeable difference, despite not being in contact with human-designed antibiotics, the bacteria are meeting plenty of bacterial-based antibiotics, and adapting to use them for food.

The big question of course is Will it Spread? Around the quarters of the isolated strains belonged to orders containing clinically relevant strains such as Salmonella and E. coli, meaning that hypothetically at least antibiotic consumption should be able to spread. On the other hand, actual consumption of antibiotics is unlikely to provide a greater evolutionary advantage than just resistance, and will confer a larger metabolic load on the bacteria. Although the pathways of antibiotic metabolism have not yet been fully determined, the first few steps seem to be similar to well-known resistance mechanisms (particularly in penicillin consumption). One conclusion, therefore, is that only part of the metabolic pathway would be (or already has been) passed on to pathogenic organisms, enough to provide resistance without placing unnecessary metabolic burdens on the cell.

Hat tip to Byte Size Biology for alerting me to the paper.

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Dantas, G., Sommer, M., Oluwasegun, R., & Church, G. (2008). Bacteria Subsisting on Antibiotics Science, 320 (5872), 100-103 DOI: 10.1126/science.1155157

Changing Projects

My synthetic biology project has pretty much ended now, bar the handover. I've got a little more lab work to do (still have one more restriction site in the MelA gene, and I'll have a bit to do when my vio DNA arrives) but the majority of work in that direction is over...I now have a week of safety talks to prepare me for my next project: back safely in the field of bacteria-antibiotic interactions.

I've enjoyed this project. It's been fun, I've got to meet new people, and I've learnt a lot of new and very useful techniques, particularly involved in genetic manipulation (ligation, restriction, PCR etc). I've also learnt something very important. That wherever the winding road of life may take me, it is unlikely to take me very far in the direction of synthetic biology.

It's an interesting and very exciting field, it's just not one I feel I could survive a project in. These ten weeks have been long enough, now that the novelty has worn off, I'm beginning to realise that this just isn't the area of science I'm interested in. I like exploring bacteria, how they work, what they do, how they interact with the world around them. Synthetic bacteria doesn't really cover that; it uses bacteria, sure, but only as DNA-expressing chassis for carefully constructed molecular circuits. Circuits just don't hold my interest for the length required for an in-depth project.

I can see how it could be an interesting field, for engineers becoming excited in the natural world, or biologists who suddenly realise they have a passion for circuitry and building biological machines. But not for nerdy little microbiologists who get far too excited about how bacteria behave in the worlds they inhabit, how they deal with the dangers and the changes and the constraints of the physical world.

I can't wait to get into my new lab. A whole week of safety talks is going to be...so... irritating...

Bacterial Hunting Strategies

Like every other form of life, bacteria need nutrients to survive. In the laboratory, these can be provided on agar plates at the perfect balance for growth and propagation. In the wild, however, nutrients seldom float around uneaten, which is why many bacteria have evolved to be predators, using a variety of strategies to seek out, destroy, and consume their prey: any other bacteria incapable of defending themselves.

There are a number of ways to eat other bacteria, and probably the simplest is phagocytosis, shown to the right (image from the free dictionary). It's a relatively easy system, involving nothing more than the ability to warp the cell membrane in response to binding, and release degrading enzymes once the prey has been captured. However, it does rely on the prey being smaller than you...and not forming complex multicellular structures such as biofilms, or fruiting bodies.

A second method is to parasitise, to crawl into the cell wall of your prey and destroy it from the inside out. This is the method used by the small bacterium Bdellovibrio bacteriovous, which attacks a large number of other bacteria (only Gram negatives though) and grows inside them, eventually destroying them. Its life cycle is shown below:This image is taken from the Nunez Group homepage, which contains a lot more information (and some beautiful pictures) about this method of predation.

The third option for bacteria is to use chemical warfare, release a large number of cell-destroying enzymes and then eat up the debris. This is the strategy of my Streptomyces, which excrete antibiotics capable of destroying a whole range of different bacteria. It is still not totally certain whether they do this for food, or simply to remove predators or potential rivals for space, but either way it's an effective method of killing bacteria which has been exploited by the pharmaceutical industry since Flemming first marketed the idea.

The final major strategy is to hunt. Bacteria do not all exist as solitary blobs in isolation, many species are able to form semi-multicellular structures that can move together, grow together, form spore-producing fruiting bodies and, in the case of Myxococcus, hunt together. Myxococcus xanthus is the model organism for this, capable of forming large swarms of bacteria that can swarm towards prey and then destroy it.

The image on the left (taken from the first reference below) shows a single M. xanthus bacteria (the long thin bacteria highlighted with an arrow) approaching and killing a round coccus bacteria. As soon as the xanthus touches its prey, it releases hydrolytic enzymes which destroy it, producing nutrients for the xanthus to then consume. Unlike Streptomyces (which can't move) these bacteria can form large colonies, which move forward together into an area colonised by prey, forming rippling shapes which can be seen on plates. Although xanthus are perfectly capable of hunting on their own, the large rippling group allows them to disrupt structures such as biofilms, giving more access to prey.

Gathering together in large groups also allows differentiation and division of labour within the group. While consuming prey, the M. xanthus tend to form two distinct subpopulations; bacteria near to the prey will be feeders, forming the characteristic rippling pattern. Behind them, bacteria in the less-nutrient rich area will begin to aggregate and form fruiting bodies which, if conditions suddenly turn bad (i.e all the food runs out) can sporulate, ensuring survival of the population.

It's a fascinating little world; hunters, predators, parasites, and a whole world of physical challenges to get through (swimming through water for bacteria is similar to moving through treacle for a human). The pressure and challenge of surviving in such a world has produced a whole mass of different shapes, sizes and strategies, from single celled packets of explosive chemicals to larger and more complex multicellular assemblies.

However good people get at killing bacteria, other bacteria will always be able to do it better. And that is why why study them.

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Berleman JE, & Kirby JR (2009). Deciphering the hunting strategy of a bacterial wolfpack. FEMS microbiology reviews, 33 (5), 942-57 PMID: 19519767

Scientia Pro Publica - 12th Edition

Welcome to the twelfth edition of scientia pro publica (science for the public) hosted here on Lab Rat. This is a blog carnival, designed to collect some of the most interesting posts on anything scientifically minded, written for people to understand and enjoy.

There's a wide selection for this edition, ranging in size from protein molecules within the cell to giant floating piles of trash in the sea. Being a microbiologist, of course, I'm going to start with the smallest and work up...

At the level of the very small:

We have an explanation of how Ritalin works, from Scicurious, and a look at the competition faced by sperm from Kelsey. There's also a lone little physical post about how to determine the charges on sticky-tape, at A Posteriori.


At the level of the slightly bigger:

Moving up to animal-sized things; there's a review of shore birds from DC birding blog, a good scientific look at the various myths surrounding chameleon colours at Ionion Enchantment and a great exploration of urban wildlife by Reconciliation Ecology.


At the evolutionary level:

In a wonderful example of the scientific method of working we have a post from Eric Johnson discussing laboratory work that shows evidence for the breakdown of the selfish gene theory, and then another post from Bob O'Hara saying that it doesn't. There's also a post by Cubic deconstructing an article written by David Stone about what makes a 'Darwinian'.

Closer to home - at the level of people:

Technically I suppose I should have dumped humans in with the rest of the eukaryotes, but there's enough exclusive posts about them to form a separate group. Dr Shock looks at whether Salvador Dali suffered from a mental illness, while Greg Laden examines the phenomenon of phantom touches. There's also a guest post at DermMatters about the importance of clinical photos, and why it's sometimes a good idea to take your own, as well as a glimpse back into the body-snatching era (the more dubious face of clinical anatomy) by Providentia.

At the level of society:

Two posts about using science in the court: a look at the importance of forensic evidence from Suzanne Smith, and Radio Frequency Identification from Adrienna Carlson. There's also a great post from A Blog Around the Clock, looking at scientific reporting, in the specific case of a giant pile of trash floating around in the Pacific.

And finally, if you have the need for more science blogging, the Online Universities Weblog has a list of the top 100 Science Professor's Blogs.

Living without a cell wall...

A cell wall is one of the most important features bacterial cells possess. They provide a barrier against the harsh conditions of the outside world, as well as helping the cell maintain its shape and integrity. They are vital for nutrition uptake, and for cell and chromosomal division.

They are also, however, the main point of attack for other competing organisms, and for the human body when under attack. There are numerous antibiotics that direct against the cell wall. It is thought therefore that some cells have adapted to live in the body without a cell wall, their innards kept inside by merely a small lipid membrane.

But how do they survive? How do they replicate? And, most importantly, how on earth do you study them in a lab. If you take the cell wall off a bacteria under laboratory conditions it turns inside out. And then explodes. It certainly doesn't stay in any kind of workable state.

Recently though (very recently) the Center for Bacterial Cell Biology in Newcastle have found a way to grow bacteria (Bacillus subtilis to be exact) without a surrounding cell wall. The mutation is quite simple to make, and by adjusting the outside conditions to prevent the cells being damaged, they managed to grow colonies of cells with no cell wall at all, and keep them alive to study.

One of the most interesting things about these cells was their division mechanism. In normal bacterial cells, division depends on the cell wall as an anchoring point to hold the chromosomal DNA while it divides, and then control the lengthening and splitting of the cell, as shown in the diagram below (from here):

How do cells without a cell wall manage to divide? In order to find out, the group at Newcastle took little movies of their cells, following them as they grew and developed. The movie isn't in the paper, but there are a series of stills from it, showing a single cell growing and dividing, and following a very different pattern of division than usually seen in bacterial cells, or in any cells:

Image taken from reference one: link

Instead of splitting into two in an organised manner, the cell blobs out to form a long strand, which then breaks up into many little pieces, each containing a copy of the cell DNA. The usual proteins needed for organised division (in particular FtsZ) are not required, the cell is using a totally different system.

What is even more interesting, is that this looks very similar to a system proposed by Ting F. Zhu and Jack W. Szostak for how the very first forms of proto-life might divide, back when life consisted of not much but a small membrane with a twisted DNA coil inside. Working totally indepentantly, their work was examining the growth and division of simple loops of lipid membrane. They would form one, and make it grow by adding micelles, little circles of membrane. They found that as they added them, the cell would eventually start elongating and, when agitated, split up into little blobs, which could then grow and divide in a very similar manner:

Image taken from reference two: link

This looks strikingly similar too the images of the dividing bacteria shown above. In both cases the membrane stretches out and then splits up again into little circles. The only change the proto-life would have to make to the physical behaviour of the membrane would be to make sure that copies of the DNA got packaged inside each little circle.

This makes the work done at the centre at Newcastle even more exciting. Not only are they developing systems to study and explore bacteria that are immune to a wide variety of antibiotics, they are also helping to explore how the earliest forms of life might have survived and propagated. This provides a glimpse into a world before even bacteria had evolved, and does being to light just how highly sophisticated and complex bacteria are, compared to their membranous blob-like ancestors.

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Leaver, M., Domínguez-Cuevas, P., Coxhead, J., Daniel, R., & Errington, J. (2009). Life without a wall or division machine in Bacillus subtilis Nature, 460 (7254), 538-538 DOI: 10.1038/nature08232

Zhu TF, & Szostak JW (2009). Coupled Growth and Division of Model Protocell Membranes. Journal of the American Chemical Society PMID: 19323552

The Importance of Fairy Stories

I was reading around the posts at ScienceBlogs, when I came across this one by Bioephemera which, while talking about a recent google-doodle took a quick jokey look at the respective merits of Hans Christian Ørsted and Hans Christian Andersen. The following quote from The Guardian about the issue was produced: "while there's nothing wrong with fairy stories, they haven't contributed much to the development of electric motors."

I couldn't resist it. Put "Discuss" on the end and it's practically an HPS (history and philosophy of science) essay. (I won't answer it in essay form, because I haven't got the time to plan out a nice long essay right now, but I will damn well be discussing it)

"While there's nothing wrong with fairy stories, they haven't contributed much to the development of electric motors." Discuss.

My main problem with this statement is that it seems to see science (and technological development, which I am lumping in the same field here) as an isolated process, remote and aloof from the rest of human life and development. Science, according to the guardian, progresses by scientific-minded people doing scientifically relating things and coming up with greater and better ways of achieving useful things, such as the electric motor. These scientists would be important and serious men (well...lets face it they probably were thinking of men) working away through detailed experimentation on serious topics, a far distance away from the whimsical and childish world of little stories.

Scientists, whatever Hollywood tries to insist, are people too. They grow up as children, hearing the same stories and tales and getting the same cultural and emotional baggage from the society around them. And science itself develops within that society, affected by it, changed by it and to a certain extent controlled by it as well. Ørsted grew up listening to the same kind of stories as Anderson, the only difference was that he didn't write them down.

In fact Hans Christian Andersen and fairy stories is a particularly bad example of things-that-do-not-affect-science, because Anderson wasn't just making these stories up. He was taking stories that were already being told. Folk-tales rather than fairy-tales, and folk-tales are crucial to human development. In a way, they are cultural development, especially in small communities where not very much writing occurs. They're how you teach your children, how you pass messages across, how you define what is acceptable and what isn't. How, in fact, you lay down the very rules and laws by which your society develops by, rules from which science is not exempt.

A few hundred years ago we had the Magician's Apprentice, adapted from a fairy-tale that makes it clear what happens if you mess with things you don't understand. The Victorian era brought forth the gothic novel Frankenstein, with a fairly similar message (among others). And now, in England, people protest against GM crops, for pretty much the same reason. I'm pretty sure there was at least one headline with the words "Frankenstein Fruit" in it. People have the stories in their heads, and stories are very powerful things to get rid of.

They told me when I was writing up my presentation for my project "make it a story". People understand stories, they understand things through stories. They develop, change, and form cultures, mostly based around stories. And science cannot be separated from the culture that surrounds it. Nor can it even remain strictly "scientific". Kekulé 'discovered' the ring structure of benzene after falling asleep and dreaming about a snake eating it's own tail. Science progresses through humans and humans progress through stories.

And, well, there's a reason the 'geeky-scientist' exists as a stereotype. We *like* fantasy, and science fiction, and other stories of other worlds. If you bring a child up telling them stories of fantastic places, and then bring them up slightly further by showing them the inside of a cell, they'll be hooked. It's a magical place, with magical rules, where everything moves and acts differently and, best of all, it really exists and you can get paid for exploring it.

On the face of it science may seem a long way from 'The Princess and the Pea' (although maybe not too far away from 'The Emperor's New Clothes'...) But these are the stories that western scientists and western science have grown up on, taking them, using them, being influenced by them. Without the stories, without the cultural background and development, the electric motor would have been a lot longer in arriving, and the rest of science would have been far, far slower. You cannot separate development into "that achieved by surrounding culture" and "that achieved by scientific and technological development", they're all far too tangled up in each other for that too be possible.

[There's even a book about physics and philosophy called 'The Emperor's New Mind'. You can't take the stories out of people.]

Probably more a 'graphic novelette'

I was reading through all the bioephemera archives that I missed through being hideously busy and was motivated to find something pretty and artistic connected with Synthetic biology (if my lovely gene designs weren't pretty enough...) Somewhere at the beginning of our course we were sent a link to a quick comic explaining synthetic biology, which I didn't bother with but which the engineers in our course were incredibly pleased with, and found really useful.

I went back and took a look at it last night. And actually, it's really good:The full story can be found here.

And it's actually in a nature paper! Which means it is most definitely not just a childish 'comic' and deserves to be taken seriously, like any other graphic novel.

The Lab Rat guide to DNA Synthesis

The structure of DNA is a double helix of two sugar-phosphate backbones joined by hydrogen-bonds between nitrogenous bases, as shown below:

Image from Bioinformaticsweb.org

The letters of the DNA code come from the bases; adenine (A), thymine (T), guanine (G) and cytosine (C). They code for amino-acids, which make up proteins, in groups of threes, i.e GCC codes for alanine, GGA codes for glycine etc.

Each base, along with the associated sugar and phosphate, forms its own little subunit. Joining these together in the correct order can code for any protein you want. As a Lab Rat I don't know very much about this process, except that I send the sequence off and get back a little vial full of DNA (or a stab containing the bacteria that have my DNA held on a separate plasmid). So what I'm writing here is just what I've managed to find out about the process - it might not reflect the most up-to-date method used in the top sequencing companies, but it's a plausible way to make DNA.

There are two main types of DNA synthesis. Firstly there's small sequence oligonucleotide (aka small-bit-of-DNA) synthesis, to make primers and things. Secondly there's whole gene synthesis, which deals with larger sections of DNA. As whole gene synthesis mostly involves sticking together little bits of DNA, I'm mostly going to focus on small oligonucleotide synthesis.

The basic process involves sticking the growing DNA strand to a solid support and then just washing the next DNA base through, over and over again. This is pretty much automated nowadays, so you just program a robot to do it. The supports used are mostly either Controlled Pore Glass or macroporous polystyrene (plastic with small holes to select for size, allowing the salts and bases to be washed away before the larger DNA molecule is eluted). Both of them covalently attach to the end of the DNA chain, holding it in place as the nucleotides are washed through.

In their natural state, however, nucleotides are not very reactive, so special modified versions are used. Large bulky groups such as DMT and cyanoethyl are used to block the ends of the bases and the phosphorous linkages, to stop them reacting or participating in reactions.

The first base is then attached to the support and the DMT group (attached to the bottom of the base - the five sided ring) cleaved off with an alkaline wash. The next subunit is then activated before being added to the support. This involves adding tetrazole, which cleaves off the three big rings shown on the left, making the subunit more reactive. The activated subunit is then washed through the column, where it can react with, and bind too, the preceding base.

Once all the bases have been added in the correct order the mixture is purified, to isolate the required sequence. This is done by desalting, usually with chromatography, to produce the final product.

In case anyone was wondering, the robot/machine/computer used for synthesis looks like this:

Image taken from monash university website.

Cell wall under attack - bacterial response to antibiotics

I took a quick break away from synthetic biology and DNA synthesis research the other day, to dive back into my happy little world of antibiotic research, in preparation for my new project in October. I'll be working with Streptomyces bacteria again, which after a whole summer of E. coli I'm quite looking forward to. What I'll be doing with them is examining the response of the cell wall to antibiotics.

The bacterial cell wall is made up of glycopeptide molecules (sugars and proteins joined together) and surrounds the whole cell. Without it, bacteria swiftly loose their integrity and salt-balance across the membrane, which is why many antibiotics target the cell wall in order to kill bacteria. Both for antibiotic resistance, and for surviving conditions that could damage the cell wall, bacteria have a system of monitoring the state of the cell membrane and responding quickly to any changes.

The system that was discovered in Streptomyces coelicolor (which I'll be working on) was named the sigE system, and consisted of an operon (string of genes) encoding four genes:

SigE encodes for a sigma-factor, a protein used in bacteria to switch on certain sets of genes. The cseA codes for a cell membrane lipoprotein, possibly used in a sensor system, while cseB and C are a two-component signalling system (very common in bacteria). CseC is a sensor (a histadine protein-kinase sensor for those who are interested) while cseB is the response regulator, acting out a response when it receives a signal from cseC.

And now...the science :)

In order to test that this operon was involved in cell membrane responses to antibiotics the lab carried out a variety of experiments, all producing evidence that lead towards this conclusion. The main experiments were as follows:

1. Removing the sigE operon and placing it on a separate plasmid, that activated resistance to Kanamycin. The bacteria were then plated on agar containing antibiotics and challenged with a kanamycin disk. Cell wall attacking antibiotics induced kanamycin, whereas antibiotics that attacked (say) the ribosome didn't.
2. Keeping the sigE in its original chromosomal context, the group then challenged it with different concentrations of vancomycin (an antibiotic which attacks bacterial cell walls). They then measured the level of the sigE operon proteins being produced in the cell. Higher concentrations of vancomycin, lead to more proteins.
3. Going back to the sigE-kanamycin resistant protein, they tried knocking out the sigE promoter, effectively switching all these genes off. The effect seen previously disappeared.
4. Leaving the lab, they then did some computational work, scanning the database to see what genes the sigE sigma-factor actually switched on. They found a group of 12 genes, all of which coded for cell-wall synthesis enzymes.

All of this leads up to some pretty conclusive evidence - in case of cell wall damage, the sigE operon is switched on. The interesting thing is, is that this isn't just a response to antibiotics either. It is highly unlikely that the system is able to respond to every different cell-wall destroying antibiotic, instead, the response is triggered by cell-wall intermediates, or degradation products that signal "Help - cell wall is being destroyed!" and switch on the sigE response, which produces proteins to mend it again.

But there are still a lot of unanswered questions. What is the cseC actually sensing? What is the exact purpose of the cseA? Why produce both a sigma-factor and a heafty response pathway? Which intermediates are used for activating? And, most importantly, can we hijack this somehow to kill bacteria?

I can't wait to get to work with it :D

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Hutchings, M., Hong, H., Leibovitz, E., Sutcliffe, I., & Buttner, M. (2006). The E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA Journal of Bacteriology, 188 (20), 7222-7229 DOI: 10.1128/JB.00818-06

Hong, H., Paget, M., & Buttner, M. (2002). A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics Molecular Microbiology, 44 (5), 1199-1211 DOI: 10.1046/j.1365-2958.2002.02960.x

Jacobs C, Frère JM, & Normark S (1997). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in gram-negative bacteria. Cell, 88 (6), 823-32 PMID: 9118225

Sequencing vs. synthesis

Sequencing = the process of finding out what base-pairs a piece of DNA consists of (AAAGGGAAA etc)

Synthesis = the process of actually making the DNA from separate base pairs.

In my last post I got the two processes chronically mixed up. The links lead to a description of DNA sequencing, whereas the process I describe doing with my DNA is preparing them for DNA synthesis.

I was going to edit the post but I couldn't find any meaningful way to do so, without either deleting the information or deleting the links, both of which contain (hopefully) quite interesting science.

I will write a post about synthesis at some point.

Sequencing

A friend of mine, who actually reads my blog occasionally, was very interested in the idea of DNA sequencing, fascinated by the thought that DNA could just be created in companies and then shipped out when needed. He mentioned I should write a post on DNA sequencing.

I thought about it, and realised, with a daunting sense of dread, that actually I would have to do quite a bit of research before being able to write coherently about DNA sequencing. I know the general idea, but not enough to explain to someone who doesn't already have quite a good idea of whats going on. Luckily there already is a very clear and comprehensive explanation of it over at Genetic Interference:

Part One
Part Two

So I'll just add to the story a little by describing things from the point of view of me...the scientist actually ordering the DNA.

First I need to find out what I want. This requires a literature search. For example, when I started looking for my pigment colours, I went on a quick trawl through PubMed, looking for any genes that had been shown to produce colour in E. coli. The vio gene shown in this post is just one example, at the moment I also have a brown pigment, and (hopefully soon) two genes that make green and red as well.

The next stage is to find the actual DNA sequence. Usually it's in the PubMed paper, or in NCBI - which has a huge database of all proteins that people have registered. If it's very new research, you might have to email or phone the researchers. Once you have the DNA it's a good idea to double-check it as well...compare to homologous proteins or ones with similar domains. I used the MUSCLE comparison tool for this, simply because it's the one I'm most used to.

Once you're certain you have the right sequence for what you want, you contact a DNA synthesis company. Prices vary... as far as I can work out it varies from 20 (if you're REALLY lucky) to 50p per base pair. A smallish gene is usually about 1kilo-base pair(kb) just to give an idea of the scale of things. And the price tends to just up once you get over 1kb as well. The vio gene which we are getting for free is about 6kb long.

Then you send your sequence off to get made! You can have various options for synthesis (as I am just discovering). The codons (AAA, GGG etc) can be optimised for your organism - in the case of more than one codon (the three bases) coding for one amino acid, different organisms will prefer to use different codons. You can get restriction sites removed and added (for cutting and pasting DNA parts), and extra parts added to the gene, such as an area for the beginning of protein coding, or a degradation tag, which will cause the end product protein to break down (very useful if it's a long-living protein you want to get rid of quickly).

Larger genes get sent in bacteria, on little loops of DNA called plasmids to keep them replicating inside the bacteria. You grow your culture up, and then can extract your precious DNA from them. Small bits of DNA, like primers, just arrive as naked DNA, inside a little plastic vial, and can be made up to solution with water.

After synthesis, it's a good idea to get them sequenced as well... just to check you have the right stuff.