Tiny gels!

This is quite possibly the smallest gel I have ever run:

To put this in perspective, usual gel-running machines are big clunky plastic things about 40cm square (or a little more rectangular) with all sorts of wires and things sticking out of both ends and gels floating around in TAE buffer solution.

The swish little thing shown above was 10x20 cms. All compact, only one wire, and the agarose gels come pre-packed and all ready to run. The stain is gel-green as well, which shows up under blue light, so the gel rests on top of a blue-light box. When the gel is finished you put the orange filter on, switch on the light, and observe the bands.

And, well, look at it. It's the most stylish piece of equipment I've ever worked with. It's going to be so hard saying good bye when I move into my new lab in November.

We didn't even have to pay for it. It was a freebee kindly donated by Invitrogen for our synthetic biology project.

(In case anyone was wondering the bands are just standard DNA ladders. We dug all the old ladders out of the freezer to see if they were working still. They are...but they're kind of in the wrong range, they are for little pieces of DNA and all our genes so far are looking like they're going to be quite big.)

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!)

Creating Biobricks

One of the main features of synthetic biology is that it should work very much like Lego. Genes are acquired from a standard registry, and can be introduced into bacteria in the form of plasmids (small autonomously replicating sections of DNA). The genes are in a standard format, connected to standard 'prefix' and 'suffix' sections (which contain restriction sites - cut and paste DNA sections that connect everything together, most specifically connect the gene to the plasmid).
The BioBrick part (shown bracketed in blue) contains the actual gene. In my case, the gene for the brown pigment melanin. The little circles on either side are restriction sites, enzymes can cut the DNA at these places, allowing the BioBricks to be moved around and, if necessary, stuck together. The red brackets show the plasmid. The purple square labelled 'origin' is the origin of replication, allowing the plasmid to be copied within the bacterial cell. The green 'antibiotic resistance' box is a marker, to check whether the plasmid has entered the cell. The plasmids are put into the bacteria in a process called transformation, after which the bacteria are plated out onto antibiotic plates. Only those which contain the plasmid are able to survive.

It works well on paper. And it seems to work quite well in real life. There is even a registry of standard BioBrick parts, you can order them and mix them together to form biological engineering systems. They tend to work as well, with varying degrees of success, and if all goes well hopefully my little brown pigment plasmid will be in there as well one day. All I have to do is cut the gene out of the plasmid it's currently in, remove unwanted restriction sites, and stick it into the red-bracketed plasmid shown above.

This can all be done by PCR =D

(If I get totally stuck with it, the other option is just to get the gene synthesised and stick it straight into the plasmid. Easier...but more expensive)

This time it's personal...

Well...today was going to be the day the we started the 'wet-work' of the project, the actual laboratory procedure rather than the planning of the last few weeks. Our project involves the bacterial species E. coli (a nice safe laboratory strain) and we were planning on opening the membranes up, sticking in new DNA and seeing if they expressed it.

Except of course today I was ill.

Bacterial infection.

E. coli...

The massive irony of this is not lost on me. However I am feeling a lot better this afternoon than I did this morning, and when I hit the lab tomorrow, there will be Vengeance.

(For the sake of scientific accuracy I should probably say that I don't know for sure that it's E. coli, but I've had this infection before, and it was E. coli the last few times, so it's a relatively high probability).

Bacterial Photography

One of the things I'll be doing in my project over the next few weeks is designing a synthetic microbiological system; inserting different bits of DNA into bacteria to make them do ... well ... whatever I want really. There are a huge range of things you can get bacteria to do, and over the next ten weeks (while I'm doing my project) I'm going to try and cover one a week, just to give an idea of the scope and applications of synthetic biology, especially within bacteria.

So...for this week, bacterial photography: The picture above (image courtesy of UT/UCSF) is a coliroid, a picture taken by shining light onto a bacterial plate. This is done by putting genes that produce a black pigment under the control of a light-sensing bacteria. Bacteria in the light do not produce pigment, those in the dark do, creating a photographic image when light is shone on to a lawn of bacteria.

A more in-depth explanation of what is happening is shown in the diagram on the right (taken from this paper). The green blobs are photoreceptors; they sense light and in response they activate an intracellular protein portrayed as an orange blob (the double dotted lines are the bacterial cell membrane). This activated protein can then diffuse over to the DNA, and activate the promoter for a black pigment, which is then secreted out of the cell.


When light is present it blocks this activation process by stopping the action of the photoreceptors. Bacteria in the light will therefore not produce pigment, while those in the dark do produce pigment, leading to a darker colour on the agar plate.


The main challenge involved in this process was creating the photoreceptor. E. coli (the bacterial species used for this procedure) do not have any proteins for sensing light. Instead, a light sensing protein from a cyanobacterium was used, and held in place by fusing it to a trans-membrane protein (in the diagram above the cyanobacterium receptor is the green blob while the E. coli trans-membrane protein is the dark black line). This creates a chimeric protein, which can be put on a small circle of DNA (known as a plasmid) and inserted into the bacteria, along with the genes for the pigment. The bacteria can then be grown on a large plate, ready to be used for photography.

Genetic engineering - video

As I mentioned in the last post, I'm currently doing a two-week course in synthetic biology. Along with scientists and engineered there are also a group of design students here from London. It's been really amazing so far, seeing things from different peoples points of view, it really gives a new perspective on things. And of course it is very useful to find out just how much other disciplines are aware of what happens in moleular biology.

One of the design students (you can find her website here) made an amazing video, just exploring and explaining genetic engineering and it's uses. I particularly like the bit about classification, although the little animated bacteria is fun too. It's definately worth a watch:

The Synthetic Kingdom from Daisy Ginsberg on Vimeo.

Playing with wires

As I write, I am busy on another computer programing an Arduino board to make little lights flash on and off. Thee guy next to me has made his play Billy Jean...at double speed, which is kind of annoying and fun at the same time.

Arduino is interesting. You have the little circuit board which you wire up, and then you connect it too the computer using a USB, write a program (heh), get it to run, and, if you're lucky, a little light flashes. Or Billy Jean plays at double speed.

It's so much fun!

To put this in a little bit of context, I'm in the middle of a two week synthetic-biology course. People keep trying to get me to do programing, which is slightly disturbing. I am enjoying playing with Arduino though. Almost as much as I enjoyed constructing the bed-side tables last night :D

Carnival Time!

The latest Scientia Pro Publica blog carnival is up over at mauka to makai. It's a collection of sciencey-blog posts, with a mix of writing, video-links, and pictures, from a mix of people.There are well-known science bloggers, such as Greg Laden and GrrlScientist, as well as some lesser-known ones including your very own Lab Rat.

I'm quite pleased and proud to feature. After a slightly disappointing end of term result, and not making in past the audition stage of an online writing challenge, it's nice to know that in the Lab Rat incarnation at least I seem to be managing quite well.

Resistance without genetics - persistance in bacterial populations

Most work on bacterial resistance to antibiotics tends to start with genetics. If a bacteria is able to survive a certain antibiotic, it is assumed that it has gained a gene from somewhere (and bacteria can get genes from almost anywhere) which allows it to survive. That makes sense after all...surely two bacteria with exactly the same genetic information should react identically to antibiotics?

No. Not all the time. There is an unfortunate habit of biochemists to get too wrapped up in the genetics to think about epigenetics (control of genetic expression by proteins), especially with 'simpler' organisms like bacteria. Persistence is the property of a totally identical population of bacteria to respond differently to antibiotics, despite having identical genomes they are phenotypically (phenotype = set of observable characteristics) different.

Persistent bacteria aren't only seen in response to antibiotic's either...they can be found for many types of stress, such as heat or starvation. They've been roughly split into two different types:

• Type I persisters: form in response to stress, usually at stationary phase (i.e after the initial burst of bacterial growth)
  
• Type II persisters: are seen forming throughout the bacterial lifecycle

Monitoring the growth rates of individual cells showed that even before treatment with antibiotics, many of the persister cells had reduced growth rates. The mechanism for this is not yet clear, a few genes that may be involved have been identified, but no reason for activating them (or repressing them in non-persisters), has yet been identified.

Although these sound dangerous at first glance, the medical implications of persisters are relatively limited (probably the reason why the mechanism has not been more thoroughly studied). As they have a slower growth rate, and as there are so few of them within an overall susceptible population, they can usually be cleared relatively easily by the immune system. There are, therefore, only three main areas where they are clinically relevant: immunosuppressed patients, pathogens that have adapted to the immune system, and in niches in the body that are less available to the immune system (such as within a biofilm).

The main ecological and evolutionary implications of this are that a colony of identical bacteria can 'survive' antibiotic attack by having a few of its members able to withstand that attack. Although they will grow slower under normal conditions, their ability to withstand stressful conditions means that they can essentially recover the whole colony, without the need for a major genetic change. This is true especially of diseases such as tuberculosis, where very few bacteria are needed to re-start an infection.

One thing that would be really interesting would be to study the behaviour of persisters within biofilms. Along with swarming and quorum sensing, biofilms are an example of pseudo-multicellular bacterial behaviour, circumstances under which having a small population of cells that can regenerate the whole colony would be very useful.

---

Orit Gefen & Nathalie Q. Balaban (2009). The importance of being persistent: heterogeneity of bacterial populations under antibiotic stress FEMS Microbiology Reviews, 33 (4) : 10.1111/j.1574-6976.2008.00156.x

On the classification of blobs

The first forays into microscopy revealed a whole world of blobs, tiny microscopic organisms that were invisible to the naked eye. These went through a range of different names, from the 'animalcule' denomination given by Anton van Leeuwenheok (which he used to describe everything small he saw under the microscope, including his own sperm) through 'monera' (a more specific name for certain types of blobs, namely those that weren't eukaryotic) to 'prokaryotes', a name that still stands.

Prokeryote means, literally, 'no nucleus', and it's use allows the world of living organisms to be split up into two groups: Prokaryotes and Eukaryotes. Eukaryotes are things with a nucleus, a membrane covered partition to hold DNA, as well as many separate organelles existing within their cells, such as mitochondria, endoplasmic reticulum, the Golgi apparatus...

Prokaryotes are...uh...everything else.

Which means that the label 'prokaryote' was always waiting to fall apart. After all, they may just be blobs but there are a lot of them, and some are very different blobs. Around the 1970's people started noticing that there were a group of the prokaryotes that behaved differently, mainly through studies done by Carl Woese and George E. Fox who created classification tables based on the genetic sequences of ribosomal RNA (the part of the genome most likely to be conserved, this is often used for classification, especially of things in Deep Time). This showed that there was a distinct group of prokaryotes with a mostly separate evolutionary history (more on the mostly later) to the rest of the prokaryotes. They were originally named 'archaebacteria', and together with 'eubacteria' (true-bacteria) were put in the prokaryotes group. They were blobs without a nuclei, and that was where they belonged.

However, things started to get a bit more complicated the more people looked at archaebacteria. They weren't just a group of slightly odd bacteria, they were something else. Something different. Although their metabolic pathways are similar to bacteria, their methods of turning DNA into proteins more resembles eukaryotic processes. Their flagella (tentacle like structures used for movement) have a markedly different structure from bacterial flagella. Like bacteria, they reproduce asexually and (also like bacteria) they can share their DNA around, in fact they can also share there DNA with bacteria, which makes taxonomists tear their hair out. It's very difficult to classify something when it keeps giving its DNA away, and collecting bits from other sources.

It is proposed in the SGM journal (Society for General Microbiology-journal not available on line) that the term 'prokaryote' should be scrapped altogether. As well as being an incorrect label for a large group of organisms it also produces an incorrect evolutionary perspective. The use of the eukaryote/prokaryote terms suggests a very human based linear "One upon a time there were blobs with no nuclei and then they got nuclei and then they were better" sort of story. A more correct view is that of all three superkingdoms; bacteria, archaea and eukaryotes splitting away from each other. Eukaryotes safely packaging their DNA away, allowing a more complex system to build up, yet forfeiting the ability to share bits of DNA. The archaea and bacteria on the other hand, continued to share their genetic material, just became more selective about it as they diverged (hense the 'mostly' seperate history).

Or maybe not. It might be that the archaea/eubacteria formed a very selective group of blobs, which then split further when some developed a nucleus, while the others continued to share their DNA with the bacteria, picking up different metabolic secrets. It's hard to work out; especially given that similarities between the DNA of archaea and bacteria does not necessarily show their relatedness; it might be a gene that has remained conserved in both of them for millions of years, or it might just be one that was exchanged last week.

There a several arguments against removing the 'prokaryote' as a naming system but most of them boil down to the very multicellular-centric argument of: "but they're all just blobs!" The three superkingdoms of archaea, bacteria and eukaryote are a far more accurate, and scientifically and taxonomically correct way of looking at things than the prokaryote/eukaryote model.

My only complaint is that I spent ages in secondary school trying to learn how to spell 'prokaryote'... removing the name means I could have spent that time doing something far more useful...like building paper planes and reading 'Redwall'.