Chopping bits out of the genome

Generally bacteria genomes tend to be fairly minimal in the amount you can remove from them. Unlike eukaryotes, which can have whole swathes of genome that codes for very little, bacteria, with their limited space for a chromosome, need every gene they can get. They just don't have the space for unnecessary genes.

Streptomyces bacteria, however, have bigger genomes and the luxary to invest in genes which are not strictly necessary for bacterial survival. These are called Secondary metabolite genes (as opposed to the necessary primary metabolites) and they code for genes that form an arsenal of weapons for the Streptomyces to deploy. Most Strep are soil-based, and they need the ability to produce secondary metabolites (such as antibiotics) to fight off invading bacteria, and clear terratory to expand their growth into.

What has recently been done (very ingeniously) is to remove the secondary metabolism genes from the bacterial species Streptomyces avermitilis creating essentially an 'empty' strep bacteria, that can grow and divide but not produce any of the secretory substances that strep are known for. The researchers managed to cut an entire 1.5Mb of DNA right out of the genome - helped by the fact that all the secondary metabolite genes cluster together on one side of the chromosome.

They did this using a common molbio technique, the cre-lox system. "Lox" is an area of DNA and "cre" is the protein that very specifically cuts DNA at the Lox site - it acts like a pair of scissors. They put a Lox site on either end of the DNA that codes for secondary metabolites (using a techinque called recombination in order to attach the lox into the chromosome) along with the DNA for the Cre protein under an inducible promoter. Once the bacteria had grown, they activated Cre production, which then cut the unwanted DNA out of the bacteria. This technique was amazingly successful and is shown diagramatically below (picture from the reference):


The avimitilis now contains no secondary metabolites at all, which makes a wonderful 'empty' system to use for studying how secondary metabolites are made. Genes from other bacteria, or even some of the removed genes, can be added back in, piece at a time, to see how much of the gene is necessary for metabolite production, and which regulatory pathways are the most important. Creating an empty cell also has potential implications for biotechnology, after all when trying to produce antibiotics you want the cell working as hard as possible just to produce your product, not wasting time and resources on other metabolites!

I'm really impressed at this kind of large scale synthetic-biology. It may be an area I end up going into in the near future...

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Komatsu M, Uchiyama T, Omura S, Cane DE, & Ikeda H (2010). Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proceedings of the National Academy of Sciences of the United States of America, 107 (6), 2646-51 PMID: 20133795

Throat bacteria that destroy invaders

I did a post about a week ago, talking about the relationship between the bodies natural (commensal) bacteria and the immune system. I was quite excited therefore to find a paper (reference below) which found a specific protease enzyme that is used by commensal throat bacteria to prevent harmful biofilm formation by Staphylococcus aureus, the bacteria responsible for MRSA.

The helpful bacteria in question is Staphylococcus epidermidis which lives naturally in the throat and nasal cavity of humans. When culturing these bacteria along with the Staph aureus it was found that some epidermidis cultures were capable of destroying biofilm formation, by using the protease Esp. The diagram below shows the effects of extracted Esp on colonies of Staphylococcus aureus (image from the reference):

Figures g and j show Gram stains of the colonies, the blue dye has just stained where bacteria are present. The remaining figures show scanning electron micrographs of the colonies taken at two different levels of magnification. For those interested, the scale bar for g,h,j and k is 10um and for i and l is 1um.

To double check that this protein was having an effect within the bacteria knockout mutants were made which removed the gene from Esp from the epidermidis. These bacteria were incapable of destroying Staph aureus growth. Adding a plasmid containing the Esp gene back into the bacteria restored their ability to fight off the Staph aureus which seems fairly conclusive. Furthermore this affect also works with VRSA and MRSA; Staph aureus which are resistant to antibiotics.

Below is a diagram of the effect of actual epidermidis bacteria on Staph aureus colonies (image from the reference).
These are nasal swabs taken from volunteers who had Staph aureus infections and were given the commensal epidermidis strains to try and clear them. It can be seen that the number of staph aureus is decreasing, although some bacteria are still present after five days of treatment. That might not necessarily be a bad thing as it allows the immune system to kick in with a response, and make antibodies ready for the next potential attack.

There are several exciting things that come out of this. Firstly the use of purified Esp as a defence against MRSA biofilms has the potential to be of major importance, although there may be clinical reasons why it's not such a good idea to spray proteases all over the inside of someones nose! From a less medically-useful perspective it's a wonderful example of bacterial-colony interaction. The kind of struggle for survival that happens inside your nose is occurring for bacteria everywhere; in soil, in the water, in the air, and even in humans.

From the Staph epidermidis point of view your nasal cavity is just a great place to live (warm, safe, lots of nutrients) and it's not going to give up that kind of living environment without a fight!

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Iwase, T., Uehara, Y., Shinji, H., Tajima, A., Seo, H., Takada, K., Agata, T., & Mizunoe, Y. (2010). Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization Nature, 465 (7296), 346-349 DOI: 10.1038/nature09074

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The Gibson Assembly Song

There are no words to describe how awesome this is. You just have to watch it:



This is The Gibson Assembly song, written by the Cambridge iGEM team 2010. If you can't see it, want it in a higher resolution or just want the link to share, find it here.

The technique they are describing is the "Gibson Assembly" which is a fairly quick and painless way of joining two bits of DNA. In more sciency terms, it works by using PCR to make genes with large overlaps (40bp) at the end. You add a Master Mix to the fragments, incubate for one hour, then just transform into whatever cell you're using. For more details of how it works go here, for the recipe of the Master Mix and detailed protocol go here, and for a program to help you design Gibson Primers, go here.

The video was just made for fun, and took less than a week to put together (not counting the time to write the words!). Filming was done mostly over one day, using one camera and a slightly broken tripod, just using spaces in the lab and the gel room. The green-screen sections were done by throwing a green table cloth over some poster display boards. The music was recorded seperately, and I think each instrument was recorded seperately as well, to get the sound balance right. It was all carried out by about nine undergraduates (and one lab rat!) and massively confused most of the supervisors.

I'm already starting to see some replies and responses to this, the most popular ones seem to be variations on the "why aren't you doing any real work!?!" and "How do you have the time with all your Science!" And given that there's been a lot of kurfluffle in the blogosphere lately about science, time spent doing science and the connection of both with Passion for science I suppose I should address this...

The undergrads who made this video had been working all summer, pretty much starting two weeks after term finished. They worked weekends. They worked evenings. At the time of doing the video, they'd worked several weeks over their stipend and had long ago stopped being paid.

Now their term has started, and they are still coming in to work, in between lectures, in between their own projects. They also have to prepare a presentation and a poster over the next month, all in between their term work. And terms here are manic; eight weeks of non-stop craziness that it's hard to fit anything into.

It was just a quick few days of Fun, in between the Science. :D

I for one welcome our glowing overlords...

I've already shown you pictures of the glowing bacterial lightbulbs, but in terms of glowing products the cambridge iGEM team is going from strength to strength. To start with the most amazing, they've made an oxygen activated bacterial bubble-lamp:



(It can be found here if the above does not work)

Maybe it's not the most efficient thing to read by, but it's an impressive level of brightness and it turns off relatively (biologically speaking!) fast. It's simply a large measuring cylinder with a load of bacterial broth inside it, and a tube to blow air through. Even shaking lets enough oxygen through to start turning on the light.

Another thing they've been doing is playing around with images on 24-well plates. For those who haven't used them, 24-well plates are usually run on a plate-reader which reads samples from every well, used mostly (in our lab) for overnight assays or (as I should be doing tomorrow) as a glorified spectrometer, measuring a range of absorbances over different wavelengths.

The iGEM team are using them to make nerdy pictures. This is my favourite:

The thing I like about the 24-well-plate pictures is that their different. Painting on plates is awesome buts it's been done before a couple of times, and the glowing pixel-pictures just look new and fresh and exciting.

There's probably going to be a bit of speculation as regards this of the "but how useful is it" type. And rest assured the iGEM team are thinking of that but at the moment I'm happy to just enjoy the fact that we have glowing pixelated space invaders sitting on the bench.

Glowing pixelated space invaders!

This is why I went into science :p

On writing and blogging

I've written about many things on this blog. Bacteria, antibiotics, um, other bacteria. But one thing I haven't really covered is blogging about blogging. There is a good reason for this, lots of other people are doing it so much better and I wouldn't know where to start, apart from floundering around waving my arms about and talking about how much more I've enjoyed blogging now I'm in this whole 'network' thing.

But then Hannah went and wrote a post about it and that post gave me confidence. I might be just a recently-graduated student with limited experience of both science and science writing, but can still write. I can't write about facultys, tenure-track, post-doc-ness, or give much breadth of experience to my topics, but what I can bring, and what I hope I always will bring is a huge amount of occasionally overwhelming enthusiasm for the bacteria I love finding out about.

What's possibly a benefit to that is that I'm always starting from the point of view that I'm probably wrong. Any comments that ask pertinent questions about what I've written have me scuttling back to the literature. This blog has helped me learn, and helped me discover a new things and most importantly has kept my learning broad. I've just graduated, which means if I go on to do a PhD I will continually be narrowing down my field of vision directed towards whatever I happen to be studying. Even during my degree it was starting to happen, and yet by keeping this blog open I can learn about things like modelling virotherapy for cancer and how plants respond to iron stress despite the fact that it's not really a part of my course. It's all interesting, and I want to keep finding out about it.

As well as giving me confidence with her post Hannah also tagged me for the bloggers with substance meme:

1. Sum up your blogging motivation, philosophy and experience in exactly 10 words.

I can do that in two words: I write.

Ten words: I cannot find a way to stop myself writing. Refrigerator.

I write. I have always written. I have whole files full of masses of paper that I scribbled bad sci-fi stories on when I was ten. Any computer I've ever used will have a folder marked "non-fiction" that is usually more crammed full of things than any other folder. I have bits of fantasy story and fanfic scribbled in the margins of my lecture notes. Every time I go on holiday I usually bring some blank paper and a pen with me, rather than (or as well as) a book to read. My A-level chemistry notes have Star Wars essays covering them and I swear I used to have a school shirt with random phrases from a Harry Potter fanfiction scribbled on the cuff.

I can't ever imagine not writing.

Somewhere around second year university I decided that I should probably channel this force for good and, after finding Ed Yong's blog and realizing that it was possible to write about science online, I started writing about science. It seemed to work well, and it's been working better and better ever since. I have bloggy friends now, and a bloggy community. I tweet stuff. The writing has become something great, and I still very much enjoy doing it.

Yes that was slightly more than ten words. This is a blog-post, not a tweet.

2. Pass it on to 10 other bloggers with substance

I think everyone I'd want to pass it onto has already been tagged, but here's ten bloggers that I enjoy reading and most of whom I'm blog-friends with anyway:

Skeptic Wonder - The Protist Person

Lucas Brouwers - who blogs at Thoughtonomics about all sort of interesting stuff

C6-H12-O2 - a blogger I found recently who writes lots of nice mol-bio articles

Angry by Choice - who won me over by writing a post about a fungi that devours worms by making little traps for them

Schooner of Science - the pirate science blogger!

MolBio Research Highlight - who got the MolBio Carnival running and does awesome tweets.

Disease of the Week - A bacteriologist, a virologist, and a lot of diseases (and currently a poll...)

Oscillator - for synthetic biology goodies

Dr Isis - for all the wonderful advice on science, motherhood and other things that I might end up doing in the future

Games with Words - who makes very good points that I sometimes disagree with

(I would have added Culturing Science to the list as well, but she tagged me so I'm not sure it counts...)

That's probably the most linked post I've ever made, and also probably more information than anyone really wanted to know about me. I am a science student who likes writing, and this blog is where it all comes together as one.

Graduation!

Finally finished! After four years of stupidly intense work and crazily long holidays I've finally officially finished my degree and left my university. I'm still living in the city, and odds are I'll come back for a PhD the year after next so it wasn't exactly a tearful goodbye, but I still managed to have fairly intense Moment when I scurried out the fire exit after having gone through the requisite amount of Latin for me to walk off with the actual degree.

I can now put letters after my name!

I've got around two weeks off before I start my summer project, which I am VERY much looking forward too. What with revision and the way the course works I haven't actually been in a lab since around March, which is far too long for a Lab Rat to be out of a lab for.

I also won't technically be a "lab rat" any more. I chose the name to mean "unpaid student lab worker" (it seemed vaguely funny at the time...) and now I am a fully fledged paid scientist (albeit one who is frantically looking for a job).

There will be more bacteria-related posts coming very shortly. This is just an apology-for-lack-of-science post because what with rehearsing for graduation, getting graduated, getting rascally drunk at graduation parties and looking for wedding dresses there hasn't been all that much time for bacteria.

~Lab Rat MSci(Hons) BA CANTAB :D

Reflections on Lab Work

I've just come to the end of my project for this year. Last week was the usual flurry of tidying, organising the freezer, and making sure all my stuff is on the Lab Computer so that anyone else who wants it can use it. I tried very hard to feel some sort of nostalgia about leaving, but to be honest all I managed was a sense of supreme exhaustion and slight relief. Besides, I'll probably be nipping in an out of there to collect results pictures, protocols, etc. for my write-up.

So I thought I'd just do a quick run-through, for anyone who's interested, about my lab experiences. I've done four lab projects to date, and enjoyed every one of them. I can't even say which one I enjoyed the most, they're all special in different ways.

1) My First Project Ever was working on bacteriophages (viruses that infect bacteria). It was fairly heavily supervised, I was working with a PhD student and helping her on her project. It was probably the most fun I've ever had in a lab; there was no pressure for a big write-up (just a small report for the funding body) or excessive pressure for me to get results, and I felt very useful being able to help. It was also the first time I'd been paid for lab work, and got that amazing rush of "woah....people will give me actual money for this!"

I started this blog during that project. The entries from back then make me laugh now. :)

2) Second project was for my course, where I switched to working on Streptomyces. Again, I was working quite closely with a PhD student, but she had kids and used to have to leave the lab about midday to take care of them. By the time the first week was up we'd worked out a system; in the morning we'd go over all the work I needed to get done, and in the afternoon she'd leave and I'd get on with it. It was quite a nice step up, as I was still being supervised, but I was doing the work all by myself.

The blog kind of died in that period, because it was insanely busy and I hadn't found out about research blogging yet.

3) The third project was the synthetic biology one, which wins the award for Most Stressful Project so far. As it was meant to be student-based we (there were seven of us, only four of whom were biological scientists) were pretty much dumped in the lab and left to get on with it. We had meetings with the supervisors once a week, and we had a PhD student to help us out (for which we were incredibly grateful) but other than that it was all down to us.

Considering I was the only one in the lab who'd ever done a project before it's amazing we managed anything really! I've got a lot of blog posts from that time, mostly because I spent about four weeks in the middle of it failing to make two point mutations. This involved lots of waiting around for gels to run, PCRs to happen, and ligations to fail, during which I would happily type away at my computer. Also at some point around then I discovered researchblogging.org and completely took off with the research side of things.

4) The project I'm currently writing up. This one has been the first project I've really thought of as my own (which makes the lack of conclusive results a bit harder to take). The lab is quite small, and as the PI and post-doc were quite busy last term, I was organising experiments and procedures pretty much on my own. I'm looking forward to the write-up, especially now I've drafted it and discovered that I actually do enough to write up. I feel more personally attached to this project than any one I've ever done before. The knowledge that my little samples are going to be sitting in the freezer for a long while before anyone bothers to dig them out actually does hurt a little. The next three weeks of write-up are going to be fun though :)

I'm quite surprised and proud that I've managed to keep up regular blogging throughout the course of the project.

5) I've already organised a summer project! As usual, there will probably be only the vaguest details of what I'm actually doing on the blog, especially as this project really should lead to a paper. But stay tuned for more bacteria-related posts. I'm sort of hooked on blogging now, and it'll probably take a lot to get me to stop.

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Multicellular bacteria

Part of my NatSci course at the moment involves group supervisions, which are made up of around ten people; a mix of students and faculty (all from the Biochemistry department). About halfway through last term we had a supervision about nuclei, and one of the supervisors raised the question, "Is it possible to be multicellular without a nucleus?" (by nucleus he meant chromosomes wrapped up in a membrane, rather than the bacterial arrangement of coiled up DNA loosly attached to the outer cell membrane). His opinion, and the overall consensus that the group seemed to come too, was no, they didn't. I managed to get a few words in about bacterial hunting packs, and sporulation and things, but they weren't really convincing enough. Also I am still just a little Lab-Ratting undergrad obsessed with bacteria, the group knows this and the general opinion seems to be that I see bacteria everywhere now. Even in multicellular form :p

Which was why I was very glad to come across a paper last week that reviewed in great detail a bacteria that is multicellular beyond all reasonable doubt. They do exist, and I feel very rightous about the whole thing, even if I'm not quite brave enough to bring it back up in supervisions.

First, a brief step back to consider just what makes something multicellular. After all, just having lots of cells in close proximity is not enough, otherwise a large colony of Staph aureus, or bits of swept up dried insect would count as multicellular. True multicellularity requires the following:

• Cell-cell adhesion: the cells must be stuck together in a fairly permanent manner
• Intercellular communication: the cells must be able to signal to each other
• Cell differentiation: probably the more important point, the cells must be doing different jobs, and must depend on the surrounding cells to survive.

Can bacteria do all that? Not very many of them can, true, or there would be large multicellular bacterial 'animals' roaming the plains. But there are a number of photosynthetic bacteria are able to form truly multicellular structures, albeit rather small ones:

Those long chains are technically all one organism, a photosynthesising cyanobacteria. The outer cell wall surrounds the whole organism in one continual envelope, and fulfills the first requirement for multicellularity. The arrows point towards larger cells which fulfill the third, these cells are different from the ones surrounding them, they have differentiated to form specialised cells whose only job is to uptake nitrogen and 'fix' it into a usable form.

The reason they've done this is simple, the enzyme required to fix nitrogen (i.e turn it from a useless inorganic form into a usable organic form) does not work in the presence of oxygen, which unfortunately is needed for respiration. That's why most animals and plants can't fix nitrogen and instead rely on food sources, or surrounding soil bacteria. Bacteria respond to this problem either by becoming totally anaerobic (not using any oxygen at all), or differentiating.

(There is a third strategy, which is to become a nitrogen fixing bacteria by night, and an aerobically respiring bacteria by day, but this requires huge amounts of energy as it means that the cell has to do a complete enzyme turnover every twelve hours)

The differentiated cell is called a heterocyst. It has a thicker cell wall to stop oxygen diffusing into the cell, and all cellular processes that might produce oxygen have been removed. Once the cell has turned into a heterocyst it cannot change back again, and is completely dependant on the cells surrounding it for the products of respiration (which it cannot carry out by itself) likewise, the surrounding cells are dependant on the heterocyst for the provision of nitrogen.

In order for the bacteria to survive, both cells are therefore vital. Furthermore the patterning of these cells are vital, having three heterocysts in a row would mean the one in the middle would be deprived of energy and could die. Each heterocyst must be surrounded by normal respiring cells, and they must also be at regular intervals, to provide nitrogen for all the cells in the organism.

This is where the cell signalling comes in. Heterocysts are created when the cells are being starved for nitrogen. Low levels of nitrogen lead to the activation of a protein called NctA, which activates another protein called hetR. These two proteins both lead to more production of each other in a positive feedback loop, until the levels of both are high enough to turn on the genes that will turn the cell into a heterocyst. It also turns on two other important genes; PatA and PatS.

PatS suppresses heterocyst formation, and this diffuses out into the surrounding cells, preventing them from differentiating. The function of PatA is a little uncertain, but it is thought to stop PatS from having an effect in the heterocyst cell itself. This means that the heterocyst will be surrounded by non-differentiated cells, which can supply it with all the energy it needs, and is shown schematically below:

As well as heterocysts, individual cells can also differentiate into other structures, such as spore like cells, which in times of nutritional stress will break up and lie dormant, turning back into fully functioning cells (and replicating to produce a fully functioning organism) when conditions improve. Several cyanobacteria can also form little lines of very small cells, called hormogonia, which have a variety of interesting functions. They show gliding motility (using either pili or slime) and can produce internal gas vesicles, which makes them bob up to the surface in water. Hormogonia are also the first infection units in symbiosis. When a cyanobacteria wants to form a symbiotic relationship with (say) a plant, it sends out these little hormogonia cells to invade the plant. Like the spore-cells, these can then turn back into normal, undifferentiated cells.

I do think it's a pity that there isn't a course here purely for studying bacteria. On the other hand, it's probably better for me as a scientist to have a slightly greater range of knowledge beyond all things prokaryotic. Especially for the upcoming exams!

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Flores, E., & Herrero, A. (2009). Compartmentalized function through cell differentiation in filamentous cyanobacteria Nature Reviews Microbiology, 8 (1), 39-50 DOI: 10.1038/nrmicro2242

Experimental Lab-ratting

It's starting to approach the time of year when PhD student's need results. Especially those who work on big human-based projects which need human subjects to fill in questionnaires and act as data points. And in the Economics and Law faculties, they are willing to spend a decent part of their budget on funding such studies, given they don't have to buy much else.

It's also the time of year when I start running out of money...

So today saw me heading over to the Law faculty to carry out an experiment in decision making. Not to bore you with the details but it involved being put in an anonymous pair (i.e I didn't know who I was paired with) and being labelled either A or B. B then answered twelve IQ questions and A was given £1 for each correct question. Here's the catch, before the questions were answered, A had to decide how much to pay B for each correct answer given.

Some A's opted for very generous amounts. One person (who either has very rich parents, a very kind heart, or was determined to screw with the experiment statistics) even gave their B the whole sum of £1 for each correct answer.

I was a B.

Apparently there are some people in this world who think that 'fair' means giving someone THIRTY PENCE PER CORRECT QUESTION while walking away with sixty pence yourself for doing bugger all.

I was very, very tempted to deliberately get the answers wrong when I saw that '30' scribbled down on my sheet of paper. But I've been trained to do exams so many times I don't think I ever could purposefully answer questions wrong and, well, I kind of need the money. I mean that was why I was there. Principles of fair behaviour are all very well, but £8.50 (£3 for participating, £3.50 for my questions and £2 for guessing the number of answers I got correct) is £8.50. It would have been hard walking away with just a fiver, although kind of funny to know that whoever-it-was was only getting three pounds for being a tight-fisted... yeah.

Anonymous partner would have ended up with around £12 and can probably work out from this that I got all my answers correct. I hope they're feeling just a little guilty. Or at the very least I hope they have six kids and a mortgage or something.

Huh.

Anyway now I have to go plate out 54 samples. I'm being an experimental lab-rat again on Friday and I've been promised a tenner for that one so yay, I can eat this week :D

Plastic from bacteria

I'm on holiday at the moment, so today's post is another section from my long essay last year, about the potential uses of biorefineries. It was written for a more scientific-based audience so might be a little harder to decipher than my usual posts.

Bioplastics

Bioplastics are polyesters that accumulate intracellularly in microorganisms in storage granules. They are usually built up from hydroxyl-acyl CoA derivatives through a range of different pathways in different microorganisms. As they are both biodegradable and biocompatible they have found numerous applications within medical and surgical fields, as well as having a greater environmental advantage over petroleum based plastics. The main disadvantages of bioplastics for commercial use are their high production and recovery costs.

The most widely produced bioplastics are poly(3-hydroxybutyrate) and poly(hydroxyalkanoic acid), referred to as PHB and PHA respectively. These both contain different β-oxidation intermediates as monomers, which are enzymatically polymerised through a condensation reaction. The structure of PHA is shown below (the 'n' indicates that the section show below is repeated multiple times):The first bioplastic to be described was PHB, found in Bacillus megaterium in 1926 by Lemoigne. It is stored in polymer form in granules within the cell.In order to decrease the recovery costs of the PHB granules, several attempts have been made to produce the secreted monomers, for polymerization outside the bacterial system. This has been achieved by expressing recombinant genes in E. coli

There are a large number of PHA polymers, ninety-one of which have been fully characterised. They are produced by both Gram negative and Gram positive bacteria via at least five different metabolic pathways. The main enzyme involved in polymer formation is PHA synthase (of the α/β hydrolase family), which polymerizes the monomers by connecting the coenzyme A thioesters of one monomer to the hydroxyl groups at positions 3, 4, 5 or 6 of the acyl moiety of the second monomer. There are four classes of PHA synthase, which are distinguished by their primary structures, substrate specificity and subunit composition. PHA synthases are found on the surface of the PHA storage granules, along with other proteins, and phospholipids.

(The structure of a PHA granule is shown above, image taken from Rehm 2003)

Engineering of recombinant bacteria that are capable of producing bioplastics requires both the transfer of a functional PHA synthase enzyme (there is no evidence as yet to suggest that any post-translational modifications of the enzyme are important for its function), and the engineering of suitable substrates that provide the enzyme with suitable substrates and sufficient concentrations. While the enzyme has been successfully transferred into model organisms such as E. coli, S. cerevisiae and even some transgenic plants, the provision of substrates is a more difficult problem as it involves dealing with large numbers of interlinked metabolic pathways. Metabolic flux analysis, carried out in transgenic E. coli, has substantially increased the carbon flux towards the production of PHB without detriment to the health of the bacteria, however this form of analysis has not yet been carried out on more complex PHA polymers.

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Madison LL, & Huisman GW (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and molecular biology reviews : MMBR, 63 (1), 21-53 PMID: 10066830

Steinbuchel, A., & Valentin, H. (1995). Diversity of bacterial polyhydroxyalkanoic acids FEMS Microbiology Letters, 128 (3), 219-228 DOI: 10.1111/j.1574-6968.1995.tb07528.x

Rehm BH (2003). Polyester synthases: natural catalysts for plastics. The Biochemical journal, 376 (Pt 1), 15-33 PMID: 12954080

Bacterial evolution: negative to positive?

The most commonly used distinction in bacterial populations is that of Gram negative and Gram positive bacteria. They are named after the method used to distinguish them: the Gram stain (developed by Hans Christian Gram). Gram positive bacteria have larger peptidoglycan cell walls, and therefore retain the crystal violet stain, whereas Gram negative bacteria have two membranes with a thin peptidoglycan wall between them, do not retain the crystal violet stain, and pick up the safranin counter-stain. The practical upshot of this is that you end up squinting at small blotched shapes under the microscope, trying to work out whether they look more pink or purple:

Gram +ve on the left, Gram -ve on the right.

As well as hinting that Mr Gram was one of those people who knows what shade 'fuchsia' is, the Gram stain is also one of the most important ways of telling what kind of bacteria you're dealing with. Despite being seemingly arbitrary, the composition of the cell wall plays a major role in determining behaviour. Gram negative bacteria (small cell wall, two cell membranes, see the picture below) tend to be motile, opportunistic, and able to colonise a wider range of environments. Gram positives on the other hand (big cell wall) are not so motile, but tend to have a huge range of excretory proteins to make up for this; almost all known antibiotics come from Gram positive bacteria.

Again, Gram +ve on the left, -ve on the right. Image from soil microbiology webpage

One thing that I've never really considered before is which one of them evolved from which. I haven't done much taxonomy, and the only time I really covered bacteria (unrelated to lab work) was in my Pathology course, which didn't seem too concerned about where different types of bacteria had come from, only what they were currently up to. The few times I did vaguely think about this though, I would have gone for the positive to negative direction. After all, surely you start with one cell membrane, and move on to two.

I recently came across a paper that came to the complete opposite conclusion, and therefore was too interesting not to read. The thing about bacterial taxonomy is that a lot of the major changes to morphology took place in Deep Time, and bacteria leave precious few fossils. Bacteria (and archaea...) had somewhere in the region of over one billion years to evolve before eukaryotic-things even started to be considered. That's a lot of time to try and sort out. To put that into context, one billion years ago from now things were just about starting to think about going multicellular. No dinosaurs, no plants even; the most complex form of life was something resembling a sofa cushion.

So how to sort out what was going on in that billion years or so? There are four main ways of going about it:

• Paleontological evidence. Bacteria don't form a huge number of fossils, but they can occasionally leave some physical evidence of their presence. For example, bacteria that eat iron will leave behind little fossilised iron cases; those that eat rocks can leave microscopic drilling holes. These provide temporal evidence for changes in structure and metabolism.
• Transition analysis. This is used to polarise major changes by turning them into a simple before-or-after question, and uses comparative, developmental, and selective arguments for determining answers. For example: did legs or wings develop first? Or, in bacterial cases: Which came first, Gram negative or Gram positive?
• Congruence testing. This searches for similarities across whole evolutionary trees, enabling loss or gain of evolutionary abilities (wings, feathers, second membranes etc) to be identified and polarised. As this is a comparison of many species, it allows potential mistakes from the arguments made in transition analysis to be found.
• Sequence trees. Sequence trees are ... problematic, but at the same time indispensably useful. They are formed by taking DNA sequences from a range of organisms and then using algorithms to tell the 'relatedness' between sequences and using these 'relatedness' levels to make evolutionary trees. They tend to be biased towards your sample distribution, undirectional, unable to properly account for generation times, and go somewhat screwy when you try to introduce horizontal gene transfer. Nevertheless they were instrumental data in showing that archaea and bacteria are two very distinct super-kingdoms (and I will freely admit that most of my distrust for them occurs because I can't get the damn things to work whenever I try them)

So...using these techniques can we get a clearer idea what was happening with bacterial membranes during those 1 billion-odd years before the arrival of eukaryotes? On the face of it; positive-to-negative seems to make more sense: start with one membrane, gain a second, possibly by gene duplication.

However like many evolutionary stories, that one falls apart a little when closer examined. Because Gram positive bacteria are not simply 'one cell membrane' they also have a massive cell wall surrounding them. Developing a second cell membrane on top of that seems absurd. And then why would the cell wall shrink? And how would anything get through this suddenly developed cell membrane. Transport proteins for the outer membrane tend to form a protein structure called a beta-sheet, while those for the inner membrane form an alpha-helix. That's a whole new system of protein folding that has to evolve pretty quickly, because otherwise the bacteria will starve, nothing can get through its outer membrane (which is balancing precariously on top of the huge cell wall...)

In view of this, the schematic seen on the right starts to make a little more sense (figure taken from the reference below). 'Murein' means 'peptidoglycan cell wall' and the cytoplasm denotes the inside of the cell. In this scenario, the double-membraned proto-bacteria (which has spend the last half-a-billion years or so evolving a well adjusted double membrane system) suddenly looses the outer membrane. A very simple genetic change would lead to a massively overgrown cell wall, which would rip the outer membrane away. The cell looses all it's outer membrane porins, and signal systems, but in return gains a highly protective cell wall, which potentially allows it to survive in different niches. How these aspects are lost genetically is another matter, and the paper rather hand-waves away by saying that unused genes tend to get lost eventually. Which is true in bacteria, they have such a small genome they don't want it getting filled up with unnecessary genes, but I have a feeling genes tend to leave something behind. Even so, the question of where the now-unnecessary genes go is possibly one of the weaker parts of this arguments (to my untrained student eyes at least.).

One thing that would really support this hypothesis would be to show that Gram positive bacteria formed a 'mono-clade' i.e came from a single universal common ancestor. Unfortunately this data is proving hard to pin down, not helped by the bacterial trick of swapping DNA around with all and sundry. Another confounding factor is the sheer space of time. Trying to determine whether a range of different modern bacteria all came from the same blob several million years ago is a daunting task. You can sort of get RNA sequence trees that support the mono-cladal Gram positives, but only if you close one eye and squint, which is not generally accepted scientific practise.

I don't think I'll ever end up going into taxonomy, even of bacteria. but it does produce fascinating ways to look at the world; how it changed, how it evolved, and how it finally turned into the way it is now. Orwell wrote, fairly famously, "He who controls the past commands the future", and when you're trying to figure out how bacterial resistance works, and preferably how to stop them getting it, that phrase takes on a whole new meaning beyond the political.

(It's not a perfect quote for this post. "Understands the past" would work better. But I'm not quite pretentious enough to go trawling through the quote archives to find something better. Any suggestions would be appreciated :p )

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Cavalier-Smith T (2006). Rooting the tree of life by transition analyses. Biology direct, 1 PMID: 16834776

Second Generation Biofuels

As part of my course last year, I wrote an extended long essay concerning the use of bacteria in biorefineries. As I've had a very lazy weekend (and to celebrate crossing the hundred post mark) I've decided to reproduce some of it here. More may be forthcoming at some point, depending on the laziness of my weekends.

Second Generation Biofuels

Second generation biofuels consist of lignocellulose material, which is broken down into simple sugars via enzymatic reactions and then fermented to produce ethanol. As lignocelluloses can be found in inedible plant matter (e.g corn husks, rice stems, and wheat stalks) they have the advantage that, unlike first generation biofuels, their utilisation does not compete with food production.

The three main components of lignocelluloses material are celluloses, hemicelluloses and lignin. These cannot be fermented directly and must therefore be broken down:The pre-treatment of the biomass is necessary both to remove lignin (although effective ligninases have been found in white-rot fungi, their rate of product turnover in bacteria is still too slow to be commercially successful) and to partially break down the cellulose to allow easier digestion by microbial processes. As the pre-treatment consists of harsh chemical processes, it would be advantageous within a biorefinery to use microbes which are able to withstand high temperatures and low pHs. For example, the cloning of thermostable cellulases into Trichoderma reesei allows a higher hydrolysis temperature compared to commercial Trichoderma enzyme, reducing the energy needed to cool the system after pretreatment with steam. The ability to save energy in this way could have a large economic impact, making the biorefinery more commercially feasible.

Currently one of the most popular microorganisms for use in lignocellulose biofuel production is Clostridium thermocellum which has an optimum temperature of around 60°C and also contains a cellulosome; a multi-protein cellulose-degrading complex attached to the bacterial cell wall. Cellulosomes are found in several bacteria, both Gram negative and positive, although they can differ in their structure and organisation (particularly of the cohesins and dockerins).
As potentially the entire process of ethanol production from lignocelluloses could be carried out by the microbes within a fermentor, the use of second generation biofuels in biorefineries has generated a lot of interest. The three main economic obstacles are the high processing costs, the narrow margin between biomass and fuel prices, and the large capital investment needed to initiate a cellulosic biorefinery. This could however, be overcome by increasing the potential for the production of high-value goods alongside the biofuel, either by adding pathways for the production of oleochemicals or bioplastics to the fermenting bacteria, or by utilising the lignin. This would provide the biorefinery with a greater capital return.

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Gilbert, H. (2007). Cellulosomes: microbial nanomachines that display plasticity in quaternary structure Molecular Microbiology, 63 (6), 1568-1576 DOI: 10.1111/j.1365-2958.2007.05640.x

Blumer-Schuette, S., Kataeva, I., Westpheling, J., Adams, M., & Kelly, R. (2008). Extremely thermophilic microorganisms for biomass conversion: status and prospects Current Opinion in Biotechnology, 19 (3), 210-217 DOI: 10.1016/j.copbio.2008.04.007

Zhang, Y. (2005). Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation Proceedings of the National Academy of Sciences, 102 (20), 7321-7325 DOI: 10.1073/pnas.0408734102

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.

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)

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...

Stages of Design

At the moment, as I mentioned in my last post. I'm trying to design a gene to get synthesised. I'm using GeneDesigner from 2.0, and I now have about 100 saved copies of my gene in various incarnations.

My first design was simple enough...the operon (a set of genes one after the other) surrounded by the biobrick prefix and suffix
For this design I used the whole vio operon, including all the bits at the beginning and end that weren't part of the gene. I was scared of cutting anything out, in case we got our nice designed gene back and it didn't make any product at all.

However, when I looked at the sequence I found that the genes within the operon were out of line. Each amino acid (the blocks that make up proteins) is coded for by three bases, for example the sequence AAAGGG will make two amino acids. AAA = lysine and GGG = glysine. However on my operon this was out of alignment; instead of getting XXX / AAA / GGG / XXX, I was getting XXA / AAG / GGX

To counter this I re-designed it, putting all the genes as separate blocks and double checking that they all made the correct proteins:
The end result was a little cramped, but it meant all the proteins were being produced as displayed, along with the prefix, suffix, and a ribosome binding site at the start of the gene.

So I showed it to my supervisor. Who looked at it, and then looked at me, and then said in a very kind voice, "Why do you *need* all that stuff around the genes?"

The thing is I'm a little afraid of taking it out. Just in case there's some sort of importance to it. But in terms of genetic engineering, and further gene manipulation, it's more useful to have each gene smartly laid out, with it's own ribosome binding site. We also want to add a promoter (at the suggestion of the ever-wonderful DNA2.0) which is a kind of START site for the gene, and allows it to be turned into mRNA (which is then turned into proteins). As the operon product has some antibiotic properties (if they're expressed at too high a level they can kill the bacteria), we want it under an inducible promoter as well, so we can turn this operon on and off by adding different chemicals to the bacteria.

So here is my plan at the moment:
Each gene is preceded by a ribosome binding site (rbs and B0034 are the same thing, I just don't know how to relabel things in GeneDesigner). The operon is preceded by a promoter and the whole thing is enclosed within the suffix and prefix.

I'm showing it to my supervisor today. I really hope it's good enough, I want to get this sequenced.

(as a point of interest, these are only a subset of the different versions currently on my computer. I also have variations on all of the above with codon optimisations, restriction sites removed and added, and different/differently placed ribosome binding sites)

EDIT (added after meeting)

I showed him the design, and this time it was pointed out that I was missing a gene...

How embarrassing.

But the rest of the design was good! So it looks like this will be the final product, bar a little fiddling about with the actual sequence:

How to (almost) damage expensive equipment

Around a week ago, a couple of men came into the lab and installed a new plate reader. This is a nifty little device that can measure the both the optical density and the fluorescence of a sample over long periods of time, essentially saving you from having to take little mini-samples out and measuring them every half-hour. It's about the size of a large printer and you put the samples into little plastic wells about half a centimeter wide and a centimeter deep then load them all up and let the machine take the readings for you.

One of the things we're working on at the moment is pigments, which we want to characterise by measuring the fluorescent spectra they give off. Very easy in a plate reader. Characterising one of them yesterday, we took the cell-samples, lysed the cells, extracted the pigment with acetone and then loaded them into the wells for the plate reader.

Luckily, someone left the wells out on the bench for a while before loading them. Because when we came to load them we immediately noticed one vital and unforgotten fact...

Acetone melts plastic.

The acetone from the extraction had melted right through the bottom of the wells. If that had been in the machine the sample would have dripped through onto the lens, which is apparently *very* expensive, and put the whole machine out of commission.

ooops...

End of exams!

So exams finally finished! Going out with a bang for the final practical paper, which was evil to the point of being insane. It seemed to be designed to cover material we hadn't been taught, about half way through I seriously began to wonder whether I'd missed a few lecture courses somewhere.

Had there been a lecture on how to calculate glycolytic flux? Was there a chemistry course I'd somehow missed? Had flow cytometry been covered at some point without my knowledge?

But no. it turned out they did actually expect us to work out how to calculate glycolytic flux from first principles. In an hour. IN FINALS. AND apparently remember back to A-level chemistry.

*is dead, Jim*

Anyway now I have more time on my hands, I want to start properly getting into research blogging. And the truly interesting stuff should start next month, when I get back into the lab. =D

Revision...

...would be a lot easier if the lecturers gave better notes. Notes that didn't leave me trying to work out colour-coded protein domains printed in greyscale with four very small slides to a page surrounded by unhelpful notes which turn into random squiggles when I drift off.

It's the option lectures that are scaring me at the moment. First terms stuff I've kind of got a hold on but I'm a bit lost with the options stuff, because I wasn't revising it as I went along. The reason I wasn't revising it? Because I was simultaneously trying to do an 8am-6pm five-days-a-week lab work project with a 5000 word write-up worth (wait for it) a whole 10% of my mark.

I swear this department is trying to kill me. Why I've agreed to do another year of it I don't know. (Except that the lab work was FUN. Just mental. Fun and mental. Like me :p )

But as soon as I get out of here, I am definitely going to make a headlong dash for the microbiology department and stay there. They will probably work me just as hard and just as crazily, but at least the scenery will be different.

Monoclonal antibodies

During my pathology course last year, monoclonal antibodies were one of those things I just couldn't 'get'. It was explained to me numerous times, by increasingly more irate and disappointed looking supervisors, but every time it was re-mentioned in lectures and supervisions I would sort of stare despairingly at whatever piece of paper was in front of me thinking 'what the hell are they again'.

"Something to do with mice, and antibodies, and making them human, or something" was usually the best I could do.

So when the subject appeared yet again during this years course, I decided to finally look it up properly and work out just what was going on.

Antibodies look like this:
The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody).

The idea of monoclonal antibody therapy is to produce a large number of essentially the same antibody, that can find and potentially destroy a specific target. The idea was to produce a kind of 'magic bullet' that went through the body picking out the specifically ill parts and removing them. And antibodies are very specific, and can be targeted to lots of different proteins.

The problem with producing them is that a single B cell (antibody-producing cell) will only last a few generations before dying. Not long enough to produce the large amounts of specifically-target antibody needed for therapy. The original solution to this problem was to use a technique known as hybridoma. Individual B cells that had been grown in mice and produced antibodies that destroyed whatever target the therapy was being designed to remove were fused with immortal myeloma cell lines. The B cell could then propagate for much longer, secreting monoclonal antibodies. The main problems with this technique were that is was slow and laborious and created problems for purifying the antigen.

The most modern technique I know of (although others are being developed) is called SLAM, which stands for Selected Lymphocyte Antibody Method. B cells are isolated from mice (or rabbits, other animals can potentially be used as well) and grown in little plastic wells until they start secreting antibodies. Single B cells are then isolated, and screened for activity. The relevant antibody genes are then cloned through PCR and expressed as recombinant antibodies. This technique is a lot faster and produces high affinity antibodies from a number of species.

Monoclonal antibodies are used in various drugs currently on the market. Lymphomas (cancerous B cells) can be treated with Zevalin (R) or Bexxar (R). Apparently on 3 February 2005, the New England Journal of Medicine reported that 59% of patients with a B-cell lymphoma were disease-free 5 years after a single treatment with Bexxar.

The thing is though, I'm being taught this as a biochemist student/researcher, not as a medical researcher. Which means that I have very little idea how useful, common, or applicable most of these techniques and products are. Academic researchers and medical researchers seem to live a world apart, something that hit me particularly hard during the conference. You could almost always tell, about half way through a talk, whether the speaker was a medical or academic researcher. There doesn't seem to be a whole lot of cross-talk between them either, which is a pity because academic research does often come up with the odd useful medical application, but of course they aren't in any position to implement it.