Delirium

Unfortunately, due to illness, I have been unable to make it to the lab for 5 days. Consequently, I am a little behind on my project and have not been able to update you all on how my proteins are doing. Whilst lying at home with a fever, I thought of myself as a mother to these poor proteins; without me they don’t stand a chance! The unlucky things have been stuck in the freezer for far too long and today was my chance to transform them so they can be expressed! I actually felt guilty for taking time off and leaving my proteins to fend for themselves. I know… I must have been deliriously ill!

Luckily, I am now reasonably well enough to start work and on my first day back my supervisor showed me the results to the first NMR. Apparently it is the best result we have had yet for the C2 domain of KIBRA. However, we still got 20 peaks less than expected. When I was first told this I didn’t really know what any of it meant, but after some extensive research about NMR on Wikipedia, I began to understand a little better. Basically, for every amino acid in my protein, there should be a peak on the graph. The distance and angles between the peaks can then help us to determine the structure of the protein. In regards to this, we are re-doing our NMR but this time using a buffer with a slightly lower pH to see if this will make a difference.

Meanwhile, I’ve been repeating the entire process of making proteins but for the HACE1 protein instead of KIBRA. Remember I said I was now working on a protein involved in cancer? Well that’s HACE1! Anyway, I’m working on getting my pretty KIBRA protein gels uploaded soon!

Broth, jelly, salt and vinegar!

What would happen if I drank a whole flask of LB broth? Is it weird that I’ve always wanted to try eating the DNA and protein gels and drinking the bacterial cultures? Maybe it’s because the gels remind me of jelly, especially when they have been dyed blue. Also, the name “broth” just makes me think about winter stews and warm soups. Yummy! Hot LB broth for dinner and blue protein jelly for pudding! However, the gels do smell a bit like vinegar after de-staining. Savoury jelly with salt and vinegar… It’s starting to sound like a bad idea.

Enough of my insanities and back onto proper science! It seems that we have a little contamination. Unfortunately, even though we purified the protein using flow chromatography, other bands of protein were found along with our desired protein band on the SDS-PAGE gel. We decided to do some secondary purification, but still got some contamination! Consequently, we started again from the transformation stage and purified some more protein. This time the experiment has been successful and, although there is still a trace of contamination, we have managed to gain some pure protein. That means we are ready to start NMR!!

Soon, I may be able to visualise the structure of the protein we have spent so long to create! In the mean time, I have been set a new task! Instead of neuroscience, the subject of my new project is cancer. I’m actually quite excited about this because oncology is an area I am extremely fascinated by and consequently something I’d be interested in continuing.

I’ll post the pictures of my yummy gels soon!

Phizzwizards, Ringbellers and Whoppsies!

This week I have been mostly eating Marmite biscuits…They’re a bit weird actually, but I need the energy. Unfortunately, the smell reminds me of the big bucket of yeast extract I sometimes use to make LB medium. I realise that I keep talking about smells, but the lab really is full of a variety of disgusting scents. There are shelves upon shelves of bottles each containing a glorious stench. Reminds me of the Big Friendly Giant who, I’m sure some of you remember, bottles up dreams. The lab is full of these “phizzwizards, ringbellers and whoppsies!” Just today a chemist came in and commented on how “funky” the lab smelt.

The lab has got very crowded all of a sudden! An explosion in the chemistry lab meant that all of the chemists have moved their experiments over to our lab. When I was younger, I remember thinking that science was all about bubbling flasks of colourful chemicals that would explode and fizz over, but as I grew up I began to discover that these concoctions were mainly myths. In a way, when I was told about the explosion, my childhood excitement was almost reignited. However, this was quickly snuffed out again once I discovered that the explosion was in fact just a faulty ceiling that decided to collapse…

Something rather embarrassing happened to me the other day. As some of you may know, I can be a rather jumpy individual and I was picking up some Eppendorf tubes from a 95°C water bath, when one of the lids popped open. Instead of just jumping I let out a loud shriek! Fortunately, I was the only person in the lab at the time, but I can’t believe I was scared of a popping lid! How will I be able to continue my career in lab work if I can’t even handle an Eppendorf tube?

Oh dear. My supervisor just asked me to write a research proposal to Cancer Research! The rest of the lab team seem to think it's a good idea to let me do it... They must be mad. Oh well, I'll try my best and see how it goes!



Now for a short, scientific update! I have re-done a few experiments and finally got some clear expression of my protein (see gel below)!! Consequently, I am in the middle of purifying it at the moment. This involves selecting for my protein of interest, using a flow chromatography column that binds my protein via its His-tag, therefore purifying it. Encouragingly, there was a strong peak on the graph for my elution buffer! Hopefully this means that I have got lots of my protein and not contamination, which is what I am checking at the moment by doing yet another SDS-PAGE gel (a gel that separates proteins according to their size).

SDS-PAGE

Lanes 1, 9 + 13: Soluble fractions of the 3 different plasmids I am using.
Lanes 2, 10 + 14: Insoluble fractions of the 3 different plasmids.
Lane 3: Flow through for first plasmid
Lanes 4-7: Elution for first plasmid in order of time. Lane 5 shows the strongest expression of a protein. Black line on the diagram measures it off at the protein ladder and it seems to be the expect size.
Lane 8: Protein ladder.
Lane 11: Flow through for second plasmid
Lane 12: Elution for the second plasmid in order of time.
Lane 15: Flow through for the third plasmids
Lane 16: Elution for the third plasmid in order of time.

Chromatogram

Oh bacteria, you sly dog you!

You know that feeling you get when someone you relied on lets you down? Well, I have that feeling with my bacteria right now. I know they aren’t human but it sometimes seems as though they do things on purpose just to annoy me.

I mentioned in my previous post that yesterday was to be the day of reckoning. Actually, it was a day of uncertainty and reconsideration. My hopes were that I would get clear expression of my protein in at least one of the vectors, but instead I got another “maybe”. It seems that one of my vectors is showing expression of a protein, which we think is our protein of interest, but we can’t be sure. This is because our protein ladder¹ is unclear and consequently, we are unable to see the exact size of the protein being expressed. Therefore, we have decided to re-do the experiment yet again, this time just for the most promising vector, under slightly different conditions to help improve our yield of protein.

Aaaarrrrrrggggggghhhhhh!!!!!!! Science can be so frustrating!

1. Protein ladder = A measure to show the different protein sizes. Provides a way to determine what size your proteins are

Structural Biologists Sure Know How To Party!

After attending the event in Reading, the one word I can use to describe the South West Structural Biology Science Conference would have to be “mental”. Mental in the sense that the talks and lectures were complicated, the food was amazing, the entertainment was epic and the attending scientists were quite literally mad!

As a microbiologist and immunologist, structural biology is not my strong point and I can honestly say that during some of the talks I didn’t have a clue what the speakers were talking about. However, I was not at a complete loss because many of the PhD talks were about the structures of bacteria and their proteins. This I could relate to as I have a keen interest in pathogens, which meant that I could at least understand parts of the presentations.

I don’t think I ever fully comprehended quite how vast the subject of science was until this conference because structural biology is an area I have never really thought about; although I always knew there were many different fields to specialise in. Consequently, I have now had exposure to a completely different side of science and therefore yet another aspect of it has become of interest to me.

After the first day of talks we were all taken out for dinner. A hog roast! I ate so much pork I thought I would burst, only to then discover the stir fry and pudding (strawberries and clotted cream!). To top it all off, we had an endless supply of wine! By the time dinner was over and the dance songs started to play, I was quite merry. The rest of the night was spent dancing, drinking, eating cheesy chips and drinking tea at the accommodation after party whilst watching the PCR song on YouTube. You might think that 3hours sleep (and obviously a few other factors…) would affect my enjoyment of the next day, but it’s nothing that a free full English breakfast can’t fix! Therefore, it is safe to say that I thoroughly enjoyed the conference! Who knew that scientists were such party animals? However, let me just reassure you that the conference was incredibly educational as well as fun! I don’t want any of you to think that science is just fun and games...

Back in the lab today working with sample buffer and TEMED enzymes, both of which really stink! Hoping to get some protein expression and then all I have to do is spin down my culture samples and leave them to freeze overnight to see if I can purify any protein! Tomorrow is the day of reckoning!

Cultures, colonies, proteins and ducks

Never, I repeat, NEVER go on a night out when you know that you are working on E. coli cultures the next day! The smell is terrible!

But never mind the smell, I got colonies!! That means I must have done something right! Or terribly wrong… But I prefer to believe the former! Hopefully, these colonies¹ are bacteria expressing the plasmids² I want them to express, either that or they are bacteria which have somehow gained themselves Kanamycin and Ampicillin³ resistance and have re-ligated⁴ without taking up my plasmid... However, I am optimistic that my experiment has worked!

Anyway, so these next two paragraphs are for science geeks only, explaining, in more scientific terms, what it is that I’m actually doing and how I’ve done it. If you're not a science geek and you actually have a life, please feel free to skip the next two paragraphs!

KIBRA is an important protein in a number of different pathways, including in the kidney and the brain (KI-dney-BRA-in). As well as being linked to memory, KIBRA is associated with the Hippo pathway⁵ and with the migration of podocytes⁶ in the kidney. It is seen to interact with a number of different components from various signalling pathways. In the case of memory, KIBRA reacts with proteins and lipids from other signalling proteins. One way in which they do this is via a C2 domain, the section of the protein that I am working on. Previous studies have shown that the KIBRA C2 domain is calcium-dependent, but a naturally-occurring mutant has recently been discovered in the human KIBRA gene, where two amino acids⁷ in the C2 domain have been substituted (SNPs⁸). My job is to discover whether this C2 variant is still calcium-dependent, as well as comparing the structures of both the normal wild type⁹ and the mutant C2 domains using NMR¹⁰ and X-ray crystallography¹¹. This will help us to understand the calcium affinity¹² of KIBRA and whether it aids the function of the protein. If you want to find out any more about this then I recommend this review article: KIBRA: A new gateway to learning and memory? and for those who are really interested, check out this review: Membrane binding and subcellular targeting of C2 domains .

I’ll bring you up to speed on what I have done so far. Firstly, I amplified two different lengths of DNA coding for C2 variants using PCR and picked the shorter length as that was the only one that gave a positive result on the gel. Then I used restriction digests, ligated the DNA inserts with four different plasmid vectors with complementary sticky ends and then transformed the plasmids into dh5α cells (E. coli). Afterwards, I plated them up onto the relevant antibiotic-selection plates and left them to incubate overnight, which brings me to the colonies I mentioned earlier! Big, fat, juicy colonies! I almost danced around the lab whilst setting up for the next experiment. Who knew that bacteria could make someone so happy?! To make sure that the colonies were all positive for the plasmid inserts, I used PCR of the transformed DNA. As you will see below, I got positive results for all of the colonies I tested! All that was left was to get the transformed DNA into expression vectors.

So what all the science gobbledegook above is trying to tell you is that I am working on part of a protein called KIBRA to determine its structure and function, which will help us to understand the role KIBRA plays in memory. This is quite a slow process as I essentially have to make my own KIBRA proteins to experiment with. At the moment, I have almost reached the stage of protein expression¹², meaning that I have nearly made my very own proteins! This is probably the most difficult part of the process so far because bacteria can be temperamental and can decide not to give you any purified protein (basically, it is difficult to get stuff you can use).

All of these incubation periods allow for a bit of free time, when you can often find me sitting with the ducks by the lake or on my laptop playing Tetris... I mean doing extra research! Oh, and updating my blog of course! Therefore, I should be able to update you on how my protein expression is coming along very soon. But first, I have a science conference to go to!

Science Glossary

1. Cluster of identical/cloned cells on a plate made to promote the growth of the bacteria you wish to grow
2. A section of DNA (in this case circular) used to transport foreign DNA (our DNA inserts) into the chromosomal DNA of a bacterial cell.
3. Types of antibiotic. We use plasmids coding for antibiotic resistance to later help us to select the bacteria which have taken up the plasmids.
4. The joining of two DNA strands, sticking them together.
5. Involved in the control of organ size, growth and apoptosis (death).
6. Cells in the Bowman’s capsule in the kidneys.
7. Simple organic compounds, the 20 building blocks of protein.
8. Single-nucleotide polymorphism = a mutation where a single nucleotide within the genome varies between members of a species.
9. The naturally occurring protein.
10. Nuclear magnetic resonance (the same technique used in MRI scans).
11. A method to discover the arrangement of atoms in a crystal using X-rays.
12. The stages after DNA has been translated into polypeptide chains, which are then folded into proteins

My Summer Placement

This summer, a research lab at Bath University obligingly allowed me to come and do a summer placement with them. Furthermore, the Biochemical Society improved my situation even more when they agreed to grant me funding. I was so overwhelmed by all of these gifts that were being bestowed upon me, that I was a little anxious about starting the placement. I have to admit that I was scared the Biochemical Society had made a mistake. I've never worked in a real research lab before, especially not on my own! Wouldn't I be completely inept? Aren't they worried that I'll blow up the lab by mistake?? That I'll destroy years' worth of research???


However, by the second day I was already heading straight to the lab in the mornings and carrying out my very own PCR reactions. In addition, I was signed up (on my first day!) to attend a Structural Biology Conference at Reading University in July!

I have another confession to make... I apologise to all you science research enthusiasts out there, but I have always imagined that working in research would be a very boring job... I am beginning to realise that I may have been wrong. Admittedly, it can get tedious and sometimes a little slow and repetitive. But then most jobs can be like that at times and not many people can say that they are working on ground-breaking research and may have even found a possible cure for a deadly disease. The important thing to remember during the periods of tedium, is that what I am doing matters!

So, more about my placement. The lab I am working in is researching neuroscience, specifically the molecular mechanisms of memory. The protein we are working on is called KIBRA, which is a scaffold protein and very important for memory. KIBRA is linked to Alzheimer's disease, but unfortunately not much is known about how the protein functions. Consequently, the research being done in my placement lab is to determine the structure and function of the KIBRA protein, thus helping to understand how memory is regulated.

I will be writing regular updates (probably once a week) on how I'm getting on and to tell you what work I have been doing. So if you're interested, please subscribe! Thanks   =]