Coloured bacteria vs. Angry birds

For those who didn't catch the news when it first came out, the iGEM project I carried out in the summer of '09 has been nominated for a Design Award! The Brit Insurance Design of the Year 2011 to be precise.

I'm very excited about it, even though I don't really feel like a designer (I don't own a singlething made by Apple). We're up against some pretty intense competition, such as the Angry Birds phone game, Rock band 3 and quite a few phone apps. Still, it's pretty awesome to be nominated, and I'm looking forward to seeing all the rest of the project displays.

Our two wonderful designer-friends James King and Daisy Ginsberg have put together a great video explaining the project. It's got some great animation, and clips from a radio interview with one of my fellow lab rats, and even some pictures of the infamous Orange Liberation Front...

E. chromi from Alexandra Daisy Ginsberg on Vimeo.

Oil-eating bacteria

Oil is formed from hydrocarbons: organic compounds which consist soley of the elements hydrogen and carbon. There are many, many different types of hydrocarbons, all of varying lengths and shapes, and pretty much all of them can in some way be consumed as an energy source by bacteria.

Hydrocarbons, in both ring and chain form, taken from a mix of sources

The general rule is that the shorter and fewer rings present, the more toxic the compound to bacteria (ethanol, for example, is deadly) however there are very few hydrocarbons that some bacteria somewhere won't consume. Like all living organisms, bacteria need a carbon source for energy, and when there are none of the coventional ones around (such as glucose and lactose) many of them will start consuming the ones in oil - crude oil contains a lot of carbon, which is why we burn it for energy in the first place.

Oil-eating bacteria have been found in a huge number of locations as well - seawater, freshwater, groundwater, water containing sludge, and land environments such as silt, soil and sand. They are found in arid deserts o the Middle East (which to be fair is a good place to hang out if you consume oil) and even in ice cores from Antarctica. And rather than being found widespread within seperate species, the capacity for oil consupmtion is often found in many different species within a particular location - in the bacterial world you are more likely to share habits with your nearest neighbours than with your closest relatives.

Using these bacteria for our own purposes, however, is proving slightly more problematic. You can purchse freeze-dried hydrocarbon-degrading bacteria and sprinkle them on your oil slick like minature Captain Planets, but their abilities are currently not nearly as effective as chemical detergents, or physical removal of the oil. This is mostly because these freeze-dried bacteria are specialised lab strains that (much like several of the academics that constructed them...) are often highly unsuited to the real world outside of the safe confines of the lab. Sometimes as well the pollutant is unaccessible to the bacteria, which may be floating on the top of the water while the oil is in droplets underneath the surface.

Looking into the potential of oil-eating bacteria is encouraging though, especially after incidents like the BP oil spill earlier this year. For the oil eating bactera that were hanging around, that must have been a feast day! And even though that event has now been largely forgotten by the media, there will still be coils and dropplets of oil floating around from it, and those will be cleaned up almost exclusively by bacteria.

---

Lena Ciric (2010). A natural solution: how bacterial communities can help clean up oil spills Microbiology Today, 229-231

---
Follow me on Twitter!

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...

---

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

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

...now in red

Got back from the conference last night, absolutely shattered. I had a great time, and the presentation was really well recieved, everyone seemed to love our coloured bacteria and it was my first time doing a conference presentation to Grad students. I met some new friends, and got the chance to visit Venice on the way back.

While I was away, red happened!

I'm still holding out for the glowing green myself...

To find out more visit the Cambridge iGEM team wiki.

I have a lot of stuff to catch up on, but if anything particularly amazing has happened on the internet while I've been away (other than Bora moving house - which I know about already and wish him the best of luck) drop me a note or stick it in the comments.

Bacterial Lightbulbs

I'm off at a conference in Italy this week, where I'll be doing a presentation about the iGEM work from last year along with some of the follow-up work I've been doing this summer. I'm really looking forward to it, but it does mean that I've got things to concentrate on other than blogging at the moment. I might get the last SGM post out on the weekend but I really need to try and spend that time with my fiancé rather than my science, as I feel I've been neglecting him a bit over the last week to try and get this presentation written.

So in the mean time, have a bacterial lightbulb:

This was made by this years Cambridge iGEM team (their website can be found here) by growing bacteria on agar, mashing up the agar in a little pot and growing again overnight with liquid medium poured over. Some of the resulting bacterial/agar glowing mush was then put into this lightbulb pendant (brought off ebay) to produce a little glowing bacterial lightbulb. Even better the little clumps of agar look a bit like sort of glowing crystals, which gives it a wonderfully 'science'-y effect.

The lightbulb used DNA from Vibrio fischeri, a bacteria that lives symbiotically with squid. The team acquired plasmids containing this gene controlled by the lux operon, and simply cut away the control, meaning the gene is now constitutively expressed (in the presence of oxygen) and glows away happily without any kind of exterior control. Eventually the lightbulb above will start to fade as the bacteria die and require more nutrients, but it's been sitting in the lab for a day now and is still fairly bright.

They've gotten some wonderfully spooky pictures. I don't know if it's just my imagination, or if I'm expecting this of biological systems, but it all looks faintly green to me. Green glowing things are probably the ultimate achievement in science. As well as their current white glow, they are also starting to get some other colours too, ultimately I believe their hoping for green, blue, orange red and yellow.

I'm really impressed with all their work, considering it's the efforts of nine students over a few months. They've got another month to go before they present it at MIT in Boston, which will require frantic powerpoint slides, and (I've been lead to believe) some potential animation work.

See more photos here, visit their wiki here, and follow them on Twitter here.

iGEM - student power!

iGEM is an international competition which offers students from universities all over the world the chance to design and construct a synthetic gene system in E. coli. I took part in the competition last year, and had the most exhilarating, frustrating, difficult and interesting three months I've ever had in a lab trying to get it all together. At the end of the competition all the teams meet up in MIT for three days of the craziest and most amazing conference ever. Everyone presents their work (in several parallel sessions) and you get to meet other young undergraduates who are willing to talk excitedly and continuously about bacteria.

The current iGEM team for my university are in the lab next door to me and busy doing all the traditional iGEM stuff like playing around excitedly with ancient bits of equipment (and some nice new stuff) and deciding what their mascot is:

Mr E. Coli Head was an early favourite

There have been massive thought-storming sessions, (some involving cake) and the promising beginnings of what looks like an interesting project. They have a beautiful wiki as well (much better than our little psychedelic page!) and you can follow them on Twitter.

The post-its of failed ideas

One of the hardest parts of it, is trying to come up with what my PI called 'the pitch', the idea that is going to explain your project, why you are doing it, what it's for and (unfortunately seems to be a requirement) how it's going to save the world. The problem is that there are ~150 other teams also trying to think of a pitch, and there's quite a bit of pressure to find something new and original.

This might change as the competition gets bigger, it might become more acceptible to work on smaller ideas like "making a better assay for such and such" or "improving on the work done by a team last year". This I think would be an improvement, smaller ideas have a greater chance of actually working. Many of the smaller and less well funded teams have embraced this, and have very simple and succinct project ideas (i.e "biobrick protein A, put it into E. coli and test it").

Despite not being involved it is still fun to watch the work from the sidelines, and it means I have more time to look through what other teams are doing and to look at the process of iGEM more objectively. I might put up a few more posts about it over the summer (any time I'm too busy to go through papers probably!) but if you want to watch their progress I'd suggest following them, and watching the whole drama of lab work through undergrad-project eyes.

This is there project as well. They have help when they ask for it, and supervision when required. But mostly they've been just thrown into a lab with a load of equipment just to see what happens...

How viruses hijack cellular transport systems

Even in the world of the very small, there are significant differences in size. A eukaryote cell (i.e a human cell) for example is relatively big, in microscopic terms. Most other things that interact with the cell at the microscopic level, are far smaller than it, such as bateria, viruses and signalling molecules.

A virus isn't much more than a small capsule of proteins with a little bit of DNA inside. Once it gets inside a eukaryote cell, it's very much in the position of a small child wandering into a big city. In front of it lies the vast interior of the cell, full of reactions, enzymes, proteins scurrying too and fro, mRNA being translated, proteins being folded and other busy bustling cellular processes. Surrounding it are large organelles (larger than the virus particle!) with strange and mysterious procedures going on inside them...

That sort of view, stretching across to both horizons

From here, the virus has to make its way to the nucleus, pushing its way through the crowded and complex cellular interior without being spotted as an intruder. Fortunately it has some help here, because it's facing the same problem faced by every molecule and organelle already in the cell. Transport mechanisms are already in place so that things can move around the large intracellular space with relative ease. The viruses simply hijack these transport systems and get a free ride all the way too the nucleus.

Work on the herpes simplex virus helped to produce a model of how the viral particles move around the cell. After entering the cell through the cell surface membrane, the virus is picked up by dynein which carries it along microtubes towards the nucleus. The microtubules form a network within the cell (like train rails) which the dynein motors along (using ATP energy). This is shown pictorally below:

Dynein moves in one direction along the microtubule while kinesin moves in the other direction. Together they move molecules all around the cell.

Once at the nucleus, the viral DNA enters through the nuclear membrane and is replicated inside the nucleus (entering the nucleus is a critical step for DNA-viruses; for those viruses that contain RNA this step is not so vital). The replicated DNA then comes back out of the nucleus and is transcribed into protein in the cytoplasm, which leads to the formation of new viral particles. These new viruses then have to travel back down the microtubule (carried by kinesin) to the outer membrane of the cell where they can be released into the surrounding environment and go on to infect more cells.

One interesting question is what exactly the dynein (and kenesin) bind to on the virus cell surface. As well as being an interesting point, answering this comes with the usual funding bait that if you find how viruses move inside the cell you may be able to find ways of stopping them from moving which would leave them at a severe disadvantage. To examine this the virus was isolated and the parts of the surrounding protein coat that bound to cellular factors further separated. These separated capsid proteins were then tested for their ability to bind to mammalian intracellular proteins. They found that several of the capsid proteins could bind to important transporter molecules, and furthermore that several different transporter molecules could sometimes bind to the same capsid protein.

Drawing showing the site of attachment of the motor transport proteins to the (green) virus capsule. The other end of the motor proteins is used to move along the microtubule.

As I'm in a fairly syntheticly-biological mood, I couldn't help but notice the mention at the end of the paper that this could have implications beyond virus treatment or vaccinations. The ability to create a little molecule that the cell can carry to the nucleus could have implications for both future genetic treatments and nanotechnology. The ability to get a little capsule of treatment right to the nucleus of cells could even have the potential for treating cancer cells, as it utilizes the cells own transport mechanisms to deliver treatment to the intracellular place it is needed.

---

Kerstin Radtke, Daniela Kieneke, André Wolfstein, Kathrin Michael, Walter Steffen, Tim Scholz, Axel Karger, Beate Sodeik (2010). Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures PLoS Patholgens, 6 (7) : e1000991

---

Follow me on Twitter!

Programming bacteria for search and destroy

As iGEM season is now properly underway, I thought I'd have a look at a synthetic biology paper and found this fairly awesome one about programming bacteria to hunt out and destroy atrazine, a chemical herbicide pollutant. One of the most exciting things about this work was that it didn't just involve bacteria with the ability to remove atrazine from the environment but to actively migrate towards the chemical and then destroy it.

The chemical structure of atrazine

The bacteria are controlled using riboswitches - little RNA pieces that can bind directly to a ligand (or signal molecule) and cause a change in gene expression, changes which can include switching on or off the genes involved in cell movement. Atrazine is a good molecule to start with because as well as being a relevant pollutant it also contains plenty of N-H bonds which are good for forming hydrogen-bond interactions with RNA. As well as that it has a well-characterized breakdown pathway, all components of which have been expressed successfully in E. coli.

The first stage in creating these seek and destroy bacteria was finding RNA sequences that would bind to atrazine. This was done by attaching the atrazine to a solid support and running bits of RNA past it, to see which ones would bind. They then took these successful binders and tested for riboswitch activity, i.e whether the binding to atrazine caused a conformational change in the RNA that lead to the turning on of a gene. They did this by putting a sequence complimentary to the isolated RNA upstream of the DNA for the CheZ gene, which controls motility in E. coli and then carrying out the selection process shown below:

In the absence of atrazine the CheZ is not synthesized and the bacteria stay where they are. When atrazine is added to the plate, the bacteria start to move...

Dose-dependent assays were then done on the successful RNA sequences, to characterise the reaction and check that it actually was the atrazine levels that lead to movement rather than some other confounding factor. Sequencing and examination of the binding site also helped to characterize the riboswitch and determine how it was working. The genes for atrazine-consuming ability were then added to the bacteria that moved towards the atrazine, leading to a little search-and destroy module capable of seeking out a dangerous pollutant and removing it from the environment.

---
Sinha J, Reyes SJ, & Gallivan JP (2010). Reprogramming bacteria to seek and destroy an herbicide. Nature chemical biology, 6 (6), 464-70 PMID: 20453864

---

Follow me on Twitter!

Antibiotics and Synthetic Biology

The model for bacterial death by antibiotics was fairly simply until recently. Antibiotics work by targeting a certain area of the bacteria; beta-lactams target the cell wall, Rifamycins target RNA synthesis, tetracyclins inhibit protein synthesis etc. The theory was that by inhibiting these processes, a certain vital function within the bacteria would be stopped, leading to its death.

However due to research done by Kohanski (references below) the story is looking a bit more complicated. Looking at three different classes of antibiotics they found that no matter what the site of action, all the antibiotics induced hydroxyl radicals. This was in bactericidal drugs, which actually kill bacteria, rather than bacteristatic ones (which just prevent cell growth). They also demonstrated that this mechanism of hydroxyl radical production was the end product of a chain of reactions involving damage to the TCA cycle (aka the Krebs cycle - which is a major part of respiration) which lead to damage to iron-sulphur clusters and subsequent production of the DNA-damaging hydroxyl radicals. This is shown diagramatically below, and this first paper was covered by Jim at Mental Indigestion with some great follow-up comments and discussion.

They've recently put out a review (second reference below) of which I find the most exciting parts are the two little extra-information boxes. One of them covers drug synergy and the second covers synthetic biology, both of which I'm getting increasingly more interested in.

Drug synergy

One of the most useful things about modelling drug actions is it can help to show which drugs would work most effectively in pairs. Using two drugs together can have many potential effects; it can make the treatment more effective, sometimes is can make the treatment less effective and of course some can be dangerous for the patient. Work on drug synergy showed that aminoglycoside antibiotics (which affect RNA synthesis) become more affective when given simultaneously with B-lactam antibiotics (which lead to cell wall breakdown) as the increased cell wall breakdown helps the aminoglycosides to get inside the cell. Conversely, drugs that inhibit protein synthesis are less effective when given at the same time as drugs which inhibit DNA synthesis as making it harder to synthesise proteins from sub-optimal DNA actually makes the cell more able to survive.

These interactions will affect the dosage of drugs used during synergistic treatments, and it is hoped that using two different types of antibiotics at low doses might be more healthy for the patient, and might help to combat against antibacterial resistance to one of the drugs.

Synthetic Biology

Another interesting concept the paper brings attention too is the potential use of synthetic biology to aid in both the study and application of antibiotic-related death systems. By using synthetic genes to disrupt or alter the proposed antibiotic network novel drug targets could be discovered. If turned into a high-throughput system this would be far more useful than the current screening system which tests for a potential drugs interaction with a target, rather than the ability of this interaction to lead to cell death.

Synthetic genes can be delivered into the bacterial cell via bacteriophages. Adding a synthetic gene into a bacteriophage for bacteria cell delivery has been attempted successfully before when they were used to enhance E. coli cell death by delivering genes for proteins that disrupted the DNA-repair system within the bacteria. This allowed faster and more effective killing of the bacteria at lower doses of antibiotic.

At a time when bacteria are fast becoming resistant to even the front line drugs, research that suggests novel ways of killing bacteria can produce some very useful outcomes. Using combinations of drugs at lower concentrations, or aiding antibiotics by introducing them along with synthetic genes in bacteriophages allows an increased shelf-life of the drugs that we currently possess as well as providing potential systems to aid the discovery of new antibiotics.

---

Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, & Collins JJ (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130 (5), 797-810 PMID: 17803904

Kohanski MA, Dwyer DJ, & Collins JJ (2010). How antibiotics kill bacteria: from targets to networks. Nature reviews. Microbiology, 8 (6), 423-35 PMID: 20440275

---

Follow me on Twitter!

Craig Venter's Synthetic Genome

I'm taking a miniscule break away from revision to quickly write my thoughts about the news thatt Craig Venter has finally made a 'synthetic cell' or, as Psi Wavefunction more correctly pointed out, a synthetic genome inside a normal cell. It's quite a landmark for synthetic biology; not only has an entire genome been constructed from scratch, but it's also able to replicate and make new bacteria with the same genome.

What the researchers did was to synthesise an entire genome, that is all the DNA present in the bacterial species Mycoplasma mycoides (1.08 Mbp - mega-base-pair for anyone interested), by making lots of 6 kpb (kilo-base-pair) pieces and splicing them together in yeast. They then had to carefully get the completed genome out of the yeast, and put it into an empty (i.e containing no other DNA) M. mycoides cell. The resulting bacterial cell contained only synthetically made DNA, and was capable of surviving and replicating quite happily.

Above is a scanning electron microscope picture of the dividing cells

Probably the first thing to notice about this is despite it being pretty damn impressive, it's not exactly the creation of new life. It fact, I'm not sure I'd say it's even the creation of life, just the creation of a working genome. And despite what Richard Dawkins might think you need a lot more than just a working genome to be defined as life, especially life as complicated as a bacteria. What's been achieved here is sort of the bacterial-genome equivalent of in vitro fertilisation; the DNA has been synthetically made, but it's been put into a working bacteria, containing all the proteins, lipids and other molecules that are essential for life.

I'm certainly not putting this down, it's an amazing piece of work which makes my excitement over getting a 6kb gene synthesised over the summer seem very childish. But heralding it (or indeed condemning it) as 'Scientists create life' is a little over the top. DNA is, if anything, one of the easiest things to make in the cell, given it consists of different rearrangements of four base-pairs, all in a long string. Small bits of DNA have been synthesised for a while, but as yet, no one really has much of a clue how to synthesise bacterial cell membranes, let alone how to get them to synthesise and replicate themselves.

This is the genome that Venter built. The text in the middle shows the process in full. The little letters around the edge (BssH II etc) show sites for restriction enzymes which are used to cut the genome into little pieces for analysis.

One of the questions that always comes up whenever synthetic biology is mentioned is "how safe is it?" after all, this is a man-made genome going into a bacterial cell. Surely you could make another, more dangerous genome, and put that inside a bacteria and then use it to cause destruction, or a B-movie sci-fi plot? I suppose the risk is always there but in all reality, there are much better, cheaper and faster ways to ensure destruction happens. It took Venter's team six years to get this whole thing completed and working and while it's true that the process is only going to get faster I don't see it getting any quicker than rummaging around under the sink and coming up with enough ingredients to explode. Last summer it took around one and a half months to get my 6kb gene sequenced, and two months of work completely failing to make two very small mutations in another 2kb gene. There are people who fiddle around in their garages doing synthetic gene cloning, but there appears to be a pretty non-existent overlap with terrorist activity.

So this is a big step for genomes, but a tiny step towards a fully synthetic cell. Getting the full genome was a matter of time, patience, a large supply of base-pairs, plenty of money, and doing something clever with the base-pair methylation. Trying to make a synthetic membrane requires understanding how the things work first. Every new organism starts its life in a little cocoon of useful proteins, internal-membrane structures and little filaments which help to organise DNA expression even as the DNA controls their production and regulation. Putting new DNA into this pre-existing system is something that organisms do every time they replicate. Making the whole system from scratch is something that's never actually been done before, given that each generation has at best just tweaked the design a little from whatever the original cellular background was - probably just a quick scattering of proteins surrounded by a couple of glycolipid layers. Over the billions of years it's had to evolve, this has created a mysterious and highly complex system which would be incredibly difficult for a research to attempt to replicate.

I bet Venter's labs are trying though. They had pretty-much succeeding at creating synthetic ribosomes last time I looked.

Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, & Venter JC (2010). Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science (New York, N.Y.) PMID: 20488990

---

Follow me on Twitter!

The Impact of Impact!

I went down to London for the weekend to see an exhibition by the Royal College of Art entitled "Impact!" which was a colaboration between designers and research teams to explore the potential impacts and implications of future scientific research. I always like watching when the worlds of art and science collide, and it was a good excuse to get away from my dissertation for a while.

I've done some work with designers before (during my last summer project, I wrote about it here) and I loved it. Designers bring new ways of looking at a project; they have the ability to take science out of the lab and into the real world, while still addressing social and ethical concerns. What I saw at Impact! was the ultimate in science communication and to be honest I think it showed the reasons most people get into science in the first place. It was fun, slightly geeky (five dimensional cameras!) colourful, thought-provoking and all with a wonderful overtone of sci-fi.

The project I enjoyed most (probably because I've met the designer, and saw little sneak-peaks of of it being constructed) was "Cellularity" by James King. This explored the potential of using cell-like structures to deliver pharmaceutical products into a patient, structures that over time, and years of research became so cell-like that they begin to blur the devide between life and non-life, bringing up fundamental questions abut what life even is.

Cellularity from James King on Vimeo.

Start by considering an empty cell filled with drugs and swallowed, like a tablet. Inside the body the membrane dissolves and and drug is released, similar to chemical pills. Clearly the 'cell' (if it can even be called that) is dead. Move on, design a cell which can both produce the drug itself (from a small DNA coil inside it) and replicate itself. Is that alive - or is it merely a biological drug-dispenser?

Next stage...suggested for patients who respond to no current therapy, allow the little drug-making cells to breed within the pateint, replicating in a semi-asexual manner, so that each offspring is producing a different drug. While James indroduces 'death' as a later stage in the line denoting life from non-life I think that for pure health and safety reasons it should probably slot in here. Cells that produce drugs that could potentially harm the patient must be able to die, either by self-destruction or (as James suggests) signalling to the bodys immune system to come and take them away.

If you start giving these cells the power to sense their surroundings as well (maybe to predict the best drug to produce) you get very close to something that can be called life. It's artificial life, life designed exclusively to serve the humans that use it, but life non-the-less. At this stage, it becomes almost meaningless to talk about 'life' and 'non-life' as separate boxes, and instead they become a gradiant, a sliding scale between the living and the dead. This is something that is starting to be appreciated even now when considering things like virus's, or prions. A prion is an infectious protein element, with no DNA or cell wall yet it is capible of replicating and evolving (and consequently sticking two fingers up to Dawkins a bit). If a small piece of twisted protein has a passing claim to 'life' the definition of what life actually is starts to become somewhat hazy. And scientists have made virus's in the lab, creating what could potentially be classified as living organisms from 'dead' pieces of DNA and protein.

One thing that worries me though, how many scientists went to Impact!? I'm sure plenty of designers did, and I'm sure they got a lot out of it, I certainly did. But this is really something I think more scientists should get engaged with. Designers are fun to work with, and they're good at communication especially to a general audience. They make colourful posters, and five-dimentional photography machines, and wierd spiky machines that hang from the ceiling. They bring the excitment back into biology, they remind you why you went there in the first place.

Also I really, really want a five-dimensional camera...

---

Follow me on Twitter!

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:

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...

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.

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.

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:

Biological Engineering. Now in Colour!

As I covered in this post, one of the main aims of synthetic biology is to produce modular building blocks for biology, to design and build up systems using registered and characterised parts. One thing this allows is the use of logic gates; by turning genes into abstract 'blocks' with certain properties engineers can use them to design biological circuits.

Here's an example from my work at the moment. One of the pigments I'm looking at is violacein - a purple pigment found in marine bacteria. Four genes are used to make this pigment, the first two take a molecule of tryptophan and modify it, the third joins the two modified molecules together, and the fourth further modifies this structure. This can be shown diagrammatically:VioA, vioB, vioC and vioD are the names of the genes. This diagram also shows that there is an intermediate colour within the pathway. If vioC is knocked out (so the gene can no longer function) the cells produce cyan pigment rather than violet.

This gives two separate 'states' for the cell to be in. The engineers start getting excited about this, because vioC needn't be completely destroyed in order for cyan to be produced, it can be put under the control of a certain input system, e.g an arsenic sensor. This means that the cells will normally be cyan, but in the presence of arsenic vioC is expressed and they turn violet. Instant biosensor. Easy to use, and easy to interpret.

As well as being explained biologically this can also be represented diagrammatically:
I like this diagram, because it shows just how useful a set of four genes making two colours can be. The vioA and vioD genes act as an AND gate. Putting them both under different input systems means that you can engineer bacteria that only turn cyan when two conditions are met; e.g correct temperature and correct levels of (say) iron. The vioC is then a further AND gate. Or...if the vioC is controlled by a repressor instead of an activator, a NOT gate. The possibilities are endless...

The really great thing about this is that it provides a level of abstraction for designers and engineers to work with biological systems. They don't need to know about the gene sequence, or the biological basis of gene activators and repressors. All they have to do is work with the blocks.

(Thanks to a fellow Lab Rat for the images!)