Thursday, 26 June 2014

The speed of science

Let me tell you one thing, science isn't fast!

I have learnt that there's a lot of waiting around in science! Columns need equilibrating for hours, dialysis needs to run over night, gels take at least 40 mins to run, always with the waiting!

Fortunately I am quite a patient person and the results at the end of the waiting are well worth the wait! Most of the time ;)

Tuesday, 24 June 2014

The next steps....

After my meeting with my supervisor yesterday, I now know what my next set of experiments will be! The answer to which is unknown, so this is classed as "proper research", which is very exciting!!

The TCV coat protein dimer (shown in the diagram below) has N-terminal tails, that are very sensitive to protease digestion when exposed. So when the virus is digested with chymotrypsin, they are cleaved off.

Chymotrypsin is a serine endopeptidase, that becomes active after cleavage with another protease trypsin; this enzyme cleaves between the arginine and isoleucine (R15 and L16), causing structural modification and formation of the substrate binding site. Chymotrypsin selectively cleaves peptide bonds formed by aromatic residues (eg. tyrosine, phenylalanine), as it prefers to cleave at large hydrophobic residues.

The question I will be answering is whether when this coat protein is attached to the RNA genome, in the rp-complex, does this cleavage still occur? It is thought that the RNA will be bound to the N-terminal tails and prevent the cleavage from occurring, but who knows?! Hopefully me in a few days once my experiments are complete!

Monday, 23 June 2014

Explaining dissociation and re-assembly of TCV

Last week I was working on some experiments to dissociate and re-assemble my TCV sample. These experiments were based on the paper written by my supervisor P. G. Stockley and some of his colleagues from 1986, called Structure and Assembly of Turnip Crinkle Virus, Mechanism of Reassembly in Vitro.

This paper shows how dissociation of turnip crinkle virus at elevated pH and ionic strength produces free dimers of coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits and the minor protein species, p80 (a covalently linked coat-protein dimer). Then, after isolating the coat protein and rp-complex, changing the physiological conditions and adding these two components together allows re-assembly of the virus.
The paper also shows how assembly of the virus is selective for viral RNA when in competition experiments with heterologous RNA, and that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. But last week I just focused on the dissociation and reassembly aspect of the paper.

First of all the TCV particles were dissociated. This was done by placing the virus sample in buffer containing NaCl, Tris-HCl and EDTA, along with protease inhibitors as TCV is very sensitive to these enzymes. The virus dissociates due to the ETDA in the buffer which is an chelating agent, and removes the calcium ion that is bound in the virus capsid and causes it to fall apart. The sample was fractionated using a Sephadex G150 column, which separates samples by size. When the eluted fractions were measured using spectrophotometry the spectrum was expected to look like this;

This shows that the virus has dissociated into the rp-complex which is eluted first (the first peak) and then the capsid proteins are eluted second (the second peak). 

Next re-assembly. The fraction containing the rp-complex and the fraction containing the capsid proteins were added together and dilalysed first in a buffer with high salt and a low pH, then in a buffer with low salt and a low pH. 

The paper goes on to do other experiments with specificity of the coat protein for the RNA etc. The paper can be found here I took some electron microscopy images of my virus sample and these confirm that was experiments were successful! This week I will be moving on further into the project, so I will keep you posted on what I do next!

The first battle with the EM microscope....

Today I am heading into my third week working in the Stockley lab at the University of Leeds and I can't believe how fast the time is going! 8 weeks will have flown by in no time!!

Last Friday, I was introduced to the electron microscope. Let me tell you, using that thing is pretty hard and it takes a lot of skill and patience, which I'm finding is something that is needed in science as there is a lot of waiting for things to take their course or run. Taking this into account, I think that my first attempt at obtaining images of my TCV sample was quite successful.
I had trouble getting the images in focus and so they aren't of the best resolution. We think this is mainly because of my sample preparation technique, so I have prepared more samples this morning in order to test different staining methods to see which works best for me as each method I have read seems to use a different method.

These are a few of the images I took with the electron microscope on my first trial, although they are not of great resolution, they do confirm that my experiments these last two weeks have been successful, which is great news!!

These first two images are of my TCV sample that I purified and concentrated in my first week. The dark marks are of the expected size of around 30 nm and the edge of the virus VLP can almost be seen.

This image is from the sample of dissociated virus. There are no clear structures seen in the image; this is excepted as the TCV VLP's have come apart and are now just capsid proteins and RNA. In the left corner of the image there is a area of high concentration of salt, this may be because of the buffer the virus was dissociated in as it was high in salt and contained Tris, which isn't always ideal for imaging with the electron microscope.

These last two images that I took were from the same copper grid, but from different areas of the grid. They are both from the sample of re-assembled virus. The top image shows that the re-assembly of the TCV VLP's was a success as small circular shapes have reformed, although they are not as uniform as the previous sample of TCV VLP's but this was expected. The second image looks a little different, the shapes seen are not as circular and are most likely to be the salt stable complex that has not re-assembled with the RNA to form VLP's.

As mentioned before the images are the best quality and the contrast between the background and the sample does not allow for a lot of detail to be seen of the virus structures. Hopefully by investigating different staining techniques I will be able to obtain a better quality image. Stay tuned for the results.....

Thursday, 19 June 2014

Ready for the EM!!!

So after a long week of waiting for me TCV particles to disassemble and re-assemble, carefully changing their buffer every couple of hours making sure they were in the best environment and looking after them, now the time has finally come to see the what's been going on inside my samples.
To image my virus particles I have prepared copper grids that will be used on the Electron Microscope so I will hopefully get some pretty pictures for my virus.

To prepare the samples on the grid, first you have to activate the grids by using the glow discharger. This instrument I have found to be quite temperamental. Maybe it's my inexperience with working with it but it is taking me a while to get it to work. Whilst the instrument is activating the copper disks, it glows a lovely purple colour, which got me thinking, what does the glow discharger actually do, and how does it work? So I did some research and thought it might be interesting if I shared that with you.

The glow discharger works by creating a partial vacuum and when a high voltage is applied between the cathode and anode at each end of the chamber, the electron potential ionizes the gas within the chamber. These negatively charged ions then deposit on the carbon, giving the carbon film an overall hydrophobic surface. The carbon film is on top of the copper grid and is usually hydrophobic (water hating), which would repel your sample so the glow discharger is needed to make this film hydrophilic (water loving) and hence your sample sticks to the film.

After activating the grids and delicately pipetting the sample onto the grid, the samples are then stained with a heavy metal salt that readily absorbs electrons. This is needed as biological molecules contain mainly C, O, N and H atoms which are not very dense, and the amount of electrons they absorb is minimal compared to the intensity of the electron beam of the microscope. This staining process is called "negative staining". This is because you don't see the object itself, you see an area empty of stain surrounded by stain. The area without stain is the object of interest as the sample prevents the stain from depositing onto the carbon layer.

I thought all this was rather interesting, I think it is good to know why you are undertaking a method, rather than just doing it. So now I will be researching electron microscopy, ready for using the microscope tomorrow morning and then hopefully, if all goes to plan, I will have some lovely images of virus capsids to share!

Monday, 16 June 2014

The background info to my project

Starting my second week of my internship today and after purifying my TCV sample last week, today I start on dissociation experiments.

I thought I would give a little background to my project today, as I haven't really explained what I am working on yet.

So my research project is called "Investigating the roles of sequence-specificity and packaging signals in the assembly of ssRNA plant virus capsids". The virus I am working on is called Turnip Crinkle Virus (TCV) and is a small, single-stranded, positive sense RNA virus.
I am working in the Stockley Lab at the University of Leeds, where I am currently a student studying Biochemistry.
The Stockley laboratory have recently developed single molecule fluorescence correlation spectroscopy (smFCS) as an assay for ssRNA virus reassembly in vitro at the nanomolar correlation concentrations of genome and coat protein (CP) found in vivo. Under these conditions they observed sequence-specificity in genome encapsidation, reflecting the situation in vivo. This specificity is driven by cognate interactions between multiple coat protein subunits and specific sequence/structural motifs in the genomic RNA that were termed packaging signals (PSs). These results overturn the currently widely-held paradigm that genomic RNAs are merely passengers in an electrostatically-driven assembly process controlled solely by CPs. These findings were presented in a recent paper written by A.Borodavka et al named “A two-stage mechanism of
viral RNA compaction revealed by single molecule fluorescence” (the paper can be found here They have used SELEX against viral CPs to identify putative PSs, including three major model ssRNA plant viruses; Turnip Crinkle Virus (TCV), Cowpea Chlorotic Mosaic Virus (CCMV) and Brome Mosaic Virus (BMV). The final aptamer pools were subjected to NextGen sequencing, revealing in each case conserved aptamer motifs within a number of sequence families. These sequences are putative PSs because when the genomic sequences were aligned there were statistically significant matches to the aptamers at multiple sites throughout the genomes. The vast majority of these PSs from all three viruses have predicted structures (via MFold), which encompass a stem-loop and exhibit suggestive conserved sequence motifs. They also contain the minimal assembly sequence, where interaction with CP is thought to repress translation of the helicase/replicase proteins. Modelling suggests that novel antiviral strategies targeting PS function should inhibit assembly.

These novel antiviral strategies are of great importance as plant viruses are a major problem in both the developed and third world. With billions of dollars’ worth of crops being destroyed by viruses every year this is a major threat to supporting the
growing world population.
My project aims to explore the molecular mechanism of capsid
formation and genome packaging of three simple plant virus models with the goal of potentially
uncovering new anti-viral strategies.

So far I have purified a sample of TCV from the plant leaves in which they are grown and concentrated it. The next thing I am trying to do, is the dissociate and re-assemble the RNA from its coat protein. These experiments replicate the experiments in the paper by P.K.Sorger, P.G.Stockley and S.C.Harrison named Structure and Assembly of Turnip Crinkle Virus, Mechanism of Reassembly in Vitro. I should, if everything goes to plan get a very nice graph, similar to the ones in the paper and get some nice electron microscope images of the virus I am working on, which I will post on here once I have them.

Well today has been a pretty sciency post! You may have enjoyed it, or not, but most of my other posts will not be as full on!
Until then....!!

Tuesday, 10 June 2014

First Day on the Job

Yesterday was my first day working in the Stockley lab at the University of Leeds and it was a good one! Everyone in the lab is so lovely and welcoming and I'm feeling a bit like a professional scientist!

The day was mostly filled with making up various buffers so that I could start experiments today but it was very enjoyable and I couldn't wait to start again this morning.

Today I did some column purification of my TCV sample, which is the main aim of this week. The sample that I am starting with is quite impure so I am purifying it in order to make it suitable to work with.

I realise that I haven't told you much about the project and what I am aiming to do, so I shall aim to write a more detailed post this weekend to explain.

Until next time, I am going back to "fighting the forefront of science", as one of my lecturers said to me yesterday.

Emily :)