Thursday, 27 February 2014

Rich Roberts Part III

In the final part of my interview with Nobel Laureate, Rich Roberts, I ask Rich to share his thoughts on issues that currently concern him. Rich has not only made significant advances in Molecular Biology (and he hasn't even mention his group's discovery of Base Flipping in DNA in the early 1990s!), but he has also been a passionate champion of a number of social injustices and campaigns that have, or will have, a wide impact on the world in which we live. I have also been incredibly impressed by his generosity when it comes to stepping back to let students, junior colleagues and collaborators take centre stage. These characteristics have made Rich highly sought after as a speaker and adviser for many organisations world wide. Oh and in his spare time he directs research at one of the leading Life Science Companies in the USA, New England Biolabs!

Q Over the last 20 years, you have combined your research activities and commercial role at New England Biolabs, with a number of political and scientific "crusades". Indeed, recently, you have spoken in support of Global implementation of Genetically Modified Organisms. What do you think needs to be done in this respect and why?

GMOs are incredibly important for the future of mankind because they will allow us to feed the ever-increasing population of this earth.  Without them even more people in developing countries will go hungry or starve to death than do so already. However, the developed world, which has no direct need of them personally, because their food needs are being met, have used them as a political ploy to gain power.  I am thinking specifically of Greenpeace, which has taken the political line of being opposed to them so they can save the population of Europe from their potentially “harmful effects“. What better cause for a political party than to save the population from harm.  However, those “harmful effects” are illusory.  Every experiment to demonstrate harm has only shown them to be safe.  This was easily predictable based on the far more dangerous possibilities that could come from traditional plant breeding.  With GMOs, one known gene at a time is transferred to a new plant, whereas traditionally, hundreds of unknown genes are transferred during crosses.  In addition traditional plant breeding techniques use radiation to induce mutations that it is hoped will confer beneficial properties to the plant.  Again whole plant mutagenesis introduces lots of unknown mutations that really may be harmful.  But the saddest part of this is that when Europeans declare something is dangerous in Europe, where it is not an absolute need, they cannot then go and tell the Africans it is OK for them.  Instead they have to continue the myth that GMOs are dangerous and deter their use in exactly those countries where they are desperately needed.

One of the most dramatic cases where this political attitude is causing major problems is the issue of Golden Rice, a GMO that has introduced the retinol, the precursor of Vitamin A, into rice grains. This strain was made in 1998, was available for distribution in 2002, but still waits regulatory approval because it is a GMO.  Somewhere between 1 and 2 million children die, or suffer severe developmental defects every year, because of a lack of Vitamin A. For most for these children a staple in their diet is rice.  By substituting Golden Rice for their normal white rice these problems could be averted. Millions have died because of this delay – which continues to this day because of Greenpeace activists.  How many must die, before the politicians in the Developed World are held accountable for crimes against humanity?

Q How important do you consider maths and computing for Life Scientists?

Mathematics has always been crucial to all scientists, but is now becoming of critical importance for biology for all of the reasons given above. In particular, statistics and some understanding of probability – especially when it concerns risk assessment should be in the arsenal of every life scientist, who in turn should be able to communicate the key ideas of risk assessment to the general public. Politicians revel in saving us from harm, but rarely have a good understanding themselves of just what is risky.  Would we have allowed cars to be developed if we had known how many people would be killed by them every year?  Despite all of the security measured at airports I can’t help but think that most is a complete waste of money.  While some of us may feel safer, personally I would prefer no security other than common sense and just take my chances. However, I do have a strong history of good luck.  I was originally booked to fly on one of the planes that hit the twin towers on 9/11, but just before the event I had to move my flight one day earlier.

Q Finally, what advice would you give to someone who is about to embark on a future in Science?

I give all young people the same advice.  Find out what you can be – or are already – passionate about and make a career of it.  Don’t worry about money because the happiest people in the world are those who get up on Monday and are exhilarated by the fact they have to go to work.  Those same people usually hate Friday afternoons, when they realize they have to wait two days before they can get back to work again.

A big thanks to Rich for his time and for his inspirational comments, I shall be interviewing Scientists from all areas over the coming year, please share these with your family and friends!

Wednesday, 26 February 2014

Rich Roberts Part II

In the second half of the interview, Nobel Laureate Rich Roberts considers the impact of Nobel Prize winning discoveries and his own contribution to Molecular Biology. The Nobel Prize is a useful shorthand for identifying some of the most significant achievements in Science, but the criteria have changed a little since they were first introduced in 1901 and the enduring impact of some awards has been greater than others. I wanted to find out from Rich how he viewed the prizes, but it should also be said that some discoveries and some Scientists have not been recognised by the Nobel Committee, sometimes because of the rules. So for example, with one recent exception, Nobel Prizes are not awarded posthumously and no more than three people can share a particular prize. 

Q Which Nobel Prize apart from your own, do you think has had the greatest impact on Life Sciences and which one on your own career?

Obviously, the structure of DNA and the genetic code have provided the foundation for molecular biology, but my own personal favourite Nobel Prize winner is Fred Sanger (left), who recently passed away. He developed three key methodologies – protein sequencing, RNA sequencing and DNA sequencing that have really revolutionized molecular biology.  He received the Nobel Prize for the first and last of these, but could easily have won a third for RNA sequencing. But just as important as his discoveries was the fact that he was a true scientist and unbelievably modest about his own accomplishments. He would often say he liked to fiddle around in the lab – but what a fiddler! He is my own role model for how a scientist should behave.

Q Who personally has made the greatest impact on your career in Science and how?

While Fred Sanger has served as an excellent role model throughout my life, several other people have had a great influence. The headmaster, Mr. Broakes of St. Stephen’s School in Bath, who fostered my love of mathematics was incredibly influential in showing me the value of logical reasoning. My father, who encouraged and facilitated my love of chemistry, despite knowing nothing about it himself, had an enormous impact.  Kazu Kurosawa, who was a postdoc with David Ollis, Professor of Organic Chemistry at Sheffield University (the Department is shown,left, with the old section to the fore and the Sir Richard Roberts Building added later) taught me a great deal of chemistry, but especially how to think about experiments and to understand how to do them successfully. I should also mention a few people who had a big influence by making my career possible.  Jack Strominger with whom I did a post-doc at Harvard provided the resources and encouragement for my work on tRNAs and he also introduced me to Tom RajBhandary at MIT, who was extraordinarily helpful.

Q What do you consider your greatest achievement to date in Science?

While the discovery of introns and exons and mRNA splicing is the work for which I won the Nobel Prize, I really feel that my work on restriction enzymes has had a tremendous influence on many areas of research. These enzymes were crucial to starting the Biotechnology Industry and have led to a cornucopia of interesting findings. They helped me get started in bioinformatics and to realize that predicting function from DNA sequence data is going to be crucial for all biological research in the future.

In the final part, I ask Rich about his current research and interests.....

First Blog Interview: Sir Richard Roberts Nobel Laureate Part 1

Professor Sir Richard Roberts FRS, Nobel Laureate, or Rich as he is known amongst the community of worldwide scientists, began his research career at the University of Sheffield as a PhD student in the Department of Chemistry. He went on to work at Harvard University, served as Deputy Director of Cold Spring Harbor Laboratories under Jim Watson, of DNA fame, where Rich and Phillip Sharp independently determined that genes of higher organisms contain introns. He left Cold Spring Harbor to become Research Director at New England Biolabs, known to many as the premier suppliers of the tools of Molecular Cloning, but to the luck few as major supporters of fundamental molecular biology of restriction and modification science. Rich was in fact the first person I ever emailed in about 1987. Here, Rich answers some questions I posed over the weekend, from his home in the Boston area of Massachusetts, USA. The interview will be posted in several parts.

Q How did your first become interested in Science and Biology in particular?

My interests in science started with an interest in mathematics fostered by the headmaster at my Junior School. He used to give me puzzles to solve, which I loved – and still do. Then I moved on to chemistry after my father bought me a child’s chemistry set for Christmas when I was about 11. That gave me a taste for hands on science and I was soon hooked.  After exhausting the “suggested” experiments I soon discovered I could make fireworks and explosives, which I loved. I figured I would become an industrial chemist, but when I discovered the thrill of discovery during the first year of my Ph.D. I knew that basic research was going to be much more fun. Although, my Ph.D. was strictly about chemistry I read a book by John Kendrew, called the “Thread of Life” that described the beginnings of molecular biology.  By the end of the book I was completely hooked and realized that molecular biology was going to be my chosen path.  I have never regretted the choice and now have combined molecular biology with mathematics and much of what I do is now called bioinformatics. Not only is it great fun, but it is crucial to the future of biology.  We have learnt how to sequence DNA very cheaply and so most of what we will know about biology is going to come from the careful interpretation of DNA sequences – bioinformatics! I am lucky that the two passions in my life, mathematics and molecular biology, are now combined and are providing an incredibly fulfilling life.

Q Was there anything you remember from school or University, good or bad that has influenced the way you have approached research during your career?

Some of this is covered above, but I would also state that my approach to research is to try and make sure that I always pursue some very straightforward projects that are guaranteed to produce publishable results, but I always have more risky projects going that may lead to new discoveries.  One of the keys to a successful research career is to constantly be on the lookout for those unusual results that are unexpected, but which tell you that something is happening that is unknown and unpredicted by current theory.  I love it when experiments don’t work, because then you know your ideas are wrong and Nature is behaving in some unexpected way – but of course one must be sure to repeat the results many times, because often the “unexpected” results come from a poorly designed experiment or because technically you did something wrong. I usually think of the latter as inadvertently spitting in the tube!

To be continued.....

Tuesday, 25 February 2014

First Y10 skills tests begin today

The need to train students in laboratory skills from school to University is a key feature of the scientific success in diagnostics, research and industry. I regularly visit expert labs learning new methods; I just spent a year at the School of Tropical Medicine doing just that, and what a pleasure and privilege it was. I have mentioned before the key role played by planning in the success of experiments and the value of failure (as commented on by the CEO of Phynexus). I would like to draw to your attention something that I am constantly asked by our partners: can you make sure students at the UTC are given a grounding in the technical skills required in a contemporary laboratory.

From the first class at Y10, where I asked you to verify the Beer Lambert law and demonstrate the relationship between visible spectroscopy and sample concentration, to the accurate dispensing of thousandths of millilitres using automatic pipettes you have come a long way. Today you will begin the assembly of your skills passport in which you will all individually be able to show any future employer, or academic interviewer, that you are proficient in the technical skills that are used in research, diagnostics and industry today. Take pride in your lab skills, it will serve you well in all aspects of your future working life, and in your hobbies! Today we will see how you can accurately pipette restriction enzyme reactions and analyse them by agarose gel electrophoresis. You have all done this before, but now I want perfect data. Good luck!

Monday, 24 February 2014

Synthetic Biology bring out the old....

Synthetic Biology has set the world of Biotech alight. According to one definition, "Synthetic biology is the design and construction of biological devices and systems for useful purpose". So what is it? Well it is partly the application of Molecular Cloning technology, pioneered by a small number of Bacterial Geneticists and Biochemistrst in the early 1970s and turned into a commercial enterprise by companies including New England Biolabs in particular in the 1980s. It is also in part the application of engineering principles to the construction of molecular components (devices) and whole cells (or Biological entities) using this technology. In a sense it is a "mature" form of Molecular Biology, in that it is supposedly based on concrete knowledge, rather than emerging knowledge. So how robust is the knowledge base? This is precisely what we are going to investigate this term in the Y12 project.

Molecular cloning in E.coli, has been a standard technique in most Molecular Biology labs since about 1985. In essence, the ingredients are a duplex of DNA encoding the gene of interest, a cloning vector (which can be a bacterial plasmid [shown under a microscope, right] or bacteriophage) a competent host strain and a means of selecting the uptake of the recombinant plasmid, created by joining, or "ligating" the DNA fragment with the cloning vector. Several important enzymes including restriction enzymes and DNA ligase (some of which are now replaced by recombinantion enzymes) are also needed. The cells then do their work by converting a few copies of the recombinant plasmid into many millions of copies.

This amplification of the DNA fragment was revolutionised further by the invention of PCR: the Polymerase Chain Reaction, in which a thermostable (heat resistant) DNA Polymerase is used in a number (around 20-30) cycles to amplify a DNA template (as described in the recent Master Class from the LGC scientist). So two strands become 4, 4 become 8 until amplification of a million fold occurs (see left). So to obtain a few micrograms of DNA, all that is needed for cloning, we only need a few femtograms of template DNA. This is why PCR is so good in forensic science work.

What are we going to attempt. One of our partners, Croda, have asked if would look at the possibility of cloning the genes that encose enzymes that synthesise short proteins, in fact so short that we called them peptides. These enzymes are found in certain strains of cyanobacteria and we are going to try and use PCR to amplify them, followed by capturing the genes in plasmid vectors and providing Croda with strains to test for their ability to produce peptides. If we get our experimental design correct, we should be able to make short peptides to order. This would be of considerable interest to teh Personal Care Sector.

Tuesday, 18 February 2014

Plus ça change, plus c'est la même chose

My old friend Walter Blackstock sent me this extract from the Journal Nature yesterday. Note the date!

From a letter to Nature, Vol 8, 1873

"I am well aware that in one way or other a smattering of at least one science, sometimes a confused jumble of several, is very commonly carried away from school. The science-classes there, though they may be wholly optional, are often also popular with the scholars. Interesting experiments, pretty specimens and amusing diagrams are exhibited, and some amount of information is communicated, even if no special interest should be awakened in the subject, and no clear mental gain should be the result. But this is far from the sort of position which, as it seems to me, science ought to hold in higher education."

So in short, we are back to where we started! It made me think of the Perkin story. Most of you will never have heard of William Henry Perkin, admittedly he had died 7 years before the outbreak of World War I. But at the age of 18 he was on his way to being possibly the most famous (certainly the most publicly acclaimed) Scientist of his time. Having gained entry into what is now Imperial College at the age of 15 (the picture on the left was taken by him at the age of 14: some similarities to the current Y10 group are apparent!). From there he learned Chemistry under the tuition of the eminent chemist Hoffman, who incidentally introduced the use of molecular models into organic chemistry teaching. Without which of course we would have no Watson and Crick (or even Linus Pauling). So what did he do? He discovered the dye, mauveine that gives us the colour mauve. This was a massive commercial success and led Perkin to temporarily abandon his studies and set about building equipment capable of industrial scale dye production.

The first dresses were a major success and soon, mauve was all the rage, being adopted by Queen Victoria and the Empress of France. Perkin and his brothers developed cost effective, large scale processes, filed patents and combined the science and business with considerable success. I think Perkin, who would later became an FRS a knight and general all round successful member of the establishment, has a number of admirable and impressive qualities and some of them certainly resonate with the UTC ethos. Anyone interested in his story can borrow the recent book on Perkin from me! After all, a young scientist with a passion for dye chemistry what's not to admire! Finally, does anyone get the significance of the Blog title? Maybe we have forgotten what our priorities should be in education, and we have to look back a little to make sure we fix the problem. Then think about what we are trying to achieve at the UTC in terms of your "fitness" for what comes next. I believe your Innovation Lab experiences will give you the edge over any conventional education and maybe we will see a few WH Perkins coming back to the UTC in the future!

Monday, 17 February 2014

Logs.... roots and trees?

Miss McKendry and I have been trying to find a way of supporting maths in the Innovation labs, ever since we plotted experimental growth curves for E.coli, grown at two temperatures. I also thought it would be helpful if you would be given some simple applied maths questions such as calculating serial dilutions and the number of cycles needed to achieve a million fold amplification of a DNA sample in a forensic experiment. It was then that Miss McKendry pointed out that logs are only taught in Y13, to maths, chemistry and physics A level students. So, I thought, time for another crusade. Students in the innovation labs will not leave the UTC without an appreciation of the value and use of exponential relationships in the analysis of experimental data.

I then started thinking about other fundamental experimental relationships in the Life Sciences and the inadequate preparation you have for University or Laboratory work. In fact, I have been talking about pH, with an assumption that this everyone from Y10 on would be familiar with the origin of this shorthand (negative log of the hydrogen ion concentration). When we come to measure rates of enzyme catalysed reactions, the Michaelis-Menten relationship (left), 
which takes the form of a rectangular hyperbola, or more complex still, the cooperative haemoglobin binding curve (right), both of which are bread and butter to experimental Biologists! Without at least an appreciation of the maths underlying these graphical methods, how can you hope to understand the mechanism of enzyme action, drug inhibition, antibody-antigen interactions, ion transport and so many other quantitative assays that are used in the laboratory every day. So we need to make sure that you all appreciate the relationship between the doubling of cells during growth, the amplification of DNA strands during PCR (the polymerase chain reaction) etc., and even if you are not one of those (small number of) students who is fascinated by the mathematical side of Science, you will not feel "exposed" when you go onto your placements and when you eventually graduate form the UTC. 

So watch this space for how we intend to improve your applied maths in the Innovation labs. We have discussed logs, I will get on to roots and trees in another Blog. I shall leave you now with two quotes, the first is from Galileo Galilei: "If I were again beginning my studies, I would follow the advice of Plato and start with mathematics". The second is from another slightly more recent, towering figure of science, who often expressed his modesty, Albert Einstein: "Do not worry about your difficulties in Mathematics. I can assure you mine are still greater." I know mine are! 

Friday, 14 February 2014

Etymology, entymology and the importance of language in Science

While we were awaiting the arrival of Sir Mark Walport the other week, I was talking to the Y10 students about the meaning of the Scientific term "Genome". It was also the first visit to the lab of our new English Teacher, Mr. Rob Harries and he pointed out how you can begin to work out the meaning of an unfamiliar term by breaking it down (deconstruction). So, think of a Book in the Bible....Genesis, a Greek word describing the origins of life on earth, sometimes referred to as the Book of Creation. 

Think of the study of ancestry, genealogy (which has become very popular since the census can be searched online). So in respect of genome, "Gen.." is the "prefix", taken from the shorter word gene, which describes a sequence of DNA bases that specifies a sequence of RNA, that may be translated into a protein, but has its roots in the "begetting" meaning of genesis. 

Then where does the second part (or suffix) "ome" come from? Again think of words that contain the suffix -ome. For example, chromosome, proteome etc. It refers to a body or mass of something. It was in 1920 that a German Botanist, Hans Winkler, combined a Greek prefix and a Latin suffix, to generate the word Genome, which is now a household word, defining the complete set of genes that are required to produce a particular organism. This hybridisation of Greek and Latin is famously shown by the modern word Television, derived from tele (Greek) meaning far, and videre (Latin): to see. I don't know about you, but I find this interplay between the language of Science and etymology very interesting and I am very much looking forward to discussing these issues in future lab sessions with Mr. Harries.

I am also amused by the use of language by geneticists, when they choose "nick-names" for the genes that they discover. While I was a post-doc in Switzerland in the 1980s, I kept hearing the term ftz gene, in connection with Drosophila development (see image on the left). I was trying to think what the f-t-z could possibly stand for when a Japanese post doc told me it stood for fushi tarazu, which in Japanese translates as "too few segments", obvious when you compare wild type and mutant larvae!
Ten years later at Sheffield, my old colleague and friend Phil Ingham introduced me to the world of hedgehog (a mutation that leads to stubby larvae in Drosophila, right), sonic hedgehog, indian hedgehog and desert hedgehog, all variations on this phenotype! These genes are crucial in the signalling pathways involved in vertebrate development, as well as flies. So we have a close link to etymology (the study of words) and entymology (the study of insects), separated by a single consonant, n. Which brings me to the email today from Mr. Harries, to celebrate Valentine's day, which includes the line: "Embroidered all with leaves of myrtle". Remember my reference to the periwinkle? The flower from which many alkaloids that have therapeutic use have been extracted: it is also known as myrtle. I shall leave you in the hands of a genius of Art and Science, as Leonardo da Vinci once wrote: "Principles for the Development of a Complete Mind: Study the science of art. Study the art of science. Develop your senses- especially learn how to see. Realise that everything connects to everything else."

A short addendum to the molecule of the month: Water

"It's a strange world of language in which skating on thin ice can get you into hot water". A nice quote from the Philadelphia journalist Franklin P. Jones and it came to mind when I read in the Journal Science this week about the interactions between water molecules and an interesting anti-freeze protein from the winter flounder.

Anti-freeze, (typically propylene glycol) as you will know, is what we add to our cars to prevent freezing in the radiator and screen wash. These are compounds that lower the freezing point of water, and since we don't often experience severe freezing in the UK, they are good enough for most purposes. Some living organisms have evolved mechanisms to survive extreme temperatures and the expression of antifreeze proteins is just one. The interesting feature of these molecules is that they are able to offer an alternative to the organised packing of water molecules in ice. The image above is actually an antifreeze protein from the Mealworm, Tenebrio molitor, our very own UTC model organism! 

The new structure (left) reported by Peter Davies' group in Canada, shows how a protein wraps itself around a core of water molecules: exactly the opposite of most proteins, whose folding relies on burying its amino acid side chains into an oily (hydrophobic) core. This sequestering of water molecules effectively blocks (or slows) the nucleation of ice crystals. This is a great example of how Nature meets the challenges of the physical properties of water in order to take advantage of its other many, life sustaining properties.

Samples and villains!

Are you absolutely certain, Mr. Spilsbury, that you took the correct tube containing the DNA extracted from the accused, Dr. Hawley Crippen, stored in a communal -80 freezer and, moreover, that it was this very sample that showed a positive match with the DNA extracted from the bottle of Scopolamine, found alongside a small piece of skin belonging to the deceased? I suggests my Lord, that the organisational and labeling practices carried out in your laboratories are at best suspect and at worst, downright irresponsible and dangerous. I therefore can see no reason why Dr. Crippen should not walk free and that all charges be dismissed forthwith! This is what went through my mind today when I was listening to the Master Class from Jenny McCormick from LGC forensics. But why?

We started the school year with, what in retrospect, was a poorly thought-through approach to the labeling and storage of samples generated in the lab. Some of the problems were caused by an assumption that you would all know how to mark samples clearly whether they be tubes, plates or lanes on gels. I soon realised however, that this is something you have never had to think about before. It is also true to say that the marker pens provided to label small Eppendorf tubes and the large numbers of samples generated by the class as a whole, exacerbated the problem. However, more importantly, I realised that this is a skill that is critical to carrying out any form of scientific work; and is in fact an important transferable skill. It therefore requires serious attention.

I have since chatted with a number of our commercial partners about organisational skills in general and they all see it as a priority. So when we get back, we will include labelling and storage as a key element in all experimental work flow planning. I will provide you with a generic template for you to customise for each experiment/project, following on from the excellent progress made over the last couple of weeks. We shall work together to develop a robust system for sample storage and tracking for the Innovation labs. All suggestions most welcome!

Monday, 10 February 2014

Molecule of the Month for February

This month we celebrate water; I like this quote from one of Hungary's (and in fact the world's) greatest Biochemists (Albert Szent-Gyorgi): "Life is water dancing to the tune of solids". Superficially, water is one of the simplest of molecules found in all living organisms, yet at the same time one of the most versatile; and a molecule that lies at the heart of Biology. As you can see from the diagram left, water in solution makes full use of its Hydrogen Bonding (1) potential, and this is an important aspect of its role in Biology. But first, where does it come from? We ingest water with our food and we produce water as a bye-product of the oxidation of nutrients. The final step in the catabolism of glucose, after glycolysis, the Krebs Cycle and Oxidative Phosphorylation (which generates energy in the form of ATP) is the reduction of oxygen to water.

"Water, water, everywhere, nor any drop to drink". We believe that life first emerged in the oceans (which are mostly water), which is where we inherited this dependency on water for the Chemistry of Life. The evolution of mechanisms to preserve and capture water in desert regions and to maintain liquid water under the ice, are remarkable adaptations that Sir David Attenborough discusses regularly in his books and documentaries on Life on Earth. I am sure you are well aware of these issues. If not, make sure you watch everything he has made! I particularly like his 10 minute radio podcasts on evolutionary anecdotes (look on the Radio 4 website).

Back to chemistry! Water molecules form a structured network owing to the non-linear arrangement of the OH bonds. This leaves water molecules
polarised and therefore able to form a Hydrogen bonded networks (left). However, these are weak interactions that can exchange: so we say that water molecules form a dynamic network of hydrogen bonded interactions: this is partly responsible for its unusual physical properties. The importance of this network is found in protein interactions with ligands: for example enzyme and substrates or antibodies and antigens. In order to appreciate this we have to consider the phenomenon of Entropy. 

Clausius. Gibbs, Boltzman and Maxwell (as well as others) are amongst the early pioneers of our understanding and formalisation of Entropy and the second law of thermodynamics. Entropy is often described a a measure of the disorder of matter or a system. The Universe tends to disorder (a little like an adolescent's bedroom or my research bench) and this is known to be energetically favourable. So, when a small substrate molecule interacts with and enzyme, it has been shown that this is often entropically favoured. Like a dog shaking off the water when it emerges from a pond, as a molecule binds to a protein, water is dispersed, giving a net, favourable Entropic change and thus promoting the interaction with the substrate.

Water not only provides the aqueous environment that solubilises cellular molecules, it also provides the foil to oily compounds that provides the cell with its plasma membrane. The favourable interactions between hydrophobic molecules and their "rejection" of water molecules, are central to the folding of proteins: where a soluble protein is polar on the surface and oily in the core. However, a cell membrane comprise polar phospholipids, that are oily in the core and polar on the outside and inside surfaces. Think about the proteins that straddle these membranes, they tend to be the inverse of the soluble proteins: they are oily on the outside and polar in the middle! They often provide a polar channel for communication between the inside and outside of cells. We call these channels, they include channels for ions and hormones, and sometimes even water: such as Aquaporin, which is found in the kidney for obvious reasons? This is a taste of the role of water in Biology, I haven't touched on water in a more global context, or indeed as a key molecule sitting in between metal ions in the active sites of enzymes, it is a truly exceptional molecule and one that underpins most aspects of the Life Sciences.

Sunday, 9 February 2014

Work Flow Diagrams as an aid to planning

If you set out to bake a loaf of bread, go on a holiday, get ready to run a marathon or revise for an examination, there is always an element of planning and organisation involved. The better prepared you are the more likely the outcome will be under your control. So if you are travelling to the USA for the first time, you will need to organise tickets, a passport, a visa, dollars or some form of credit card, accommodation, onward travel and possibly arrange tickets for the opera etc. If you want to travel to the USA at short notice, then there are a number of consequences. Without a passport, you will be unable to travel at all, unless you organise a 1 day passport, but for normal passport applications, this may take between 2-8 weeks. You will need a visa, but if you have internet access and online credit you can organise this the day before. As you can see, some things require planning and organisation. 

Last week, we decided to repeat the bio-prospecting experiment using your plant extracts, since the top agar plates were, in many cases, less than perfect, which made it very difficult to interpret your results. So, by repeating the experiment, hopefully this week we shall see an improved outcome. It was while we were starting the day in the lab, (the time I ask you all to discuss the day's lab work with your colleagues and come up with a plan), that I realised a Work Flow Diagram (WFD) might be the way to ensure you organise your experiment and in addition, provide you with a framework for noting down any observations as you go through the day's protocols. I was surprised to find that you all rushed into the experiment without any serious planning: this is a recipe for disaster!

I went through the general principles with a number of groups and realised that a Work Flow Diagram should form the first activity in the morning. As you know by now, I don't run the innovation labs like a structured class practical, in which all reagents and solutions are provided, rather you have to plan your experiment and obtain the materials. To help you with this we shall make use of the WFD in every lab session and it must be approved before you get started.

What should a good WFD look like? The diagram should include a brief title, the overall aim of the experiment(s), the sequence of steps which should include any key pre-incubations (e.g. pre-heating agar plates, thawing frozen samples, preparing fresh reagents, ensuring you have tubes ready for aliquoting samples etc.) You might construct a Table to help you organise the composition of multi-component assay mixtures (where appropriate), and you might work out the dilutions of samples in order to ensure the data that emerge cover the appropriate numerical range. 

I am keen for you to develop a style of your own, but as you will appreciate, a typical WFD will have some common features and some specific features that relate to the experiment and the materials. There should also be space on the WFD for noting observations, which may include suggestions for improvements, or steps that can be eliminated etc. It should be a live document! I will be asking you to produce a WFD in all classes, so start thinking about the way you will approach this. We will then try and migrate the best ideas to an iPAD format, in collaboration with the Studio.

Saturday, 8 February 2014

The importance of experimental failure

It is only natural for us to want to succeed in our endeavors, but sometimes failure is more valuable. As Sir Winston Churchill once commented: "Success consists of going from failure to failure without loss of enthusiasm". Replace "Success" with "Science" and you have your future as a Scientist in a nutshell! I am sure that Andy Murray would agree that this philosophy applies just as much to sport, or indeed any creative activity. Failure is simply an outcome that is unexpected and often unwanted. But it may give you an insight into your lack of appreciation of the complete set of variables associated with your experiment.

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When we set out last week to investigate the composition of the mealworm larvae, the outcome of the manual grinding and extraction was a little disappointing. I expected that grinding would release a significant amount of protein, and indeed the first centrifugation looked very promising. After centrifugation, the Falcon tubes had 4 layers (from the bottom up): exoskeleton (brown), disrupted cells (pale grey), around 30ml yellow supernatant and finally a fatty layer floating on the supernatant. Typically, a viscous yellow supernatant would be protein rich. However, looking at the samples run on SDS PAGE (right) suggests that the protein concentration is low, with one high molecular weight protein predominating (see lanes 1 and 2). Those of you who took spectral readings noted an absorbance maximum around 450nm. So whey was the "proteome" so simple and the total protein concentration so low? 

Some of you attempted to fractionate the sample using a Q Sepharose column. A brown colouration at the top of the column at low ionic strength (PBS buffer) was displaced by 1M NaCl. I thought this again looked promising, but SDS PAGE revealed a protein concentration too low to easily detect by Coomassie Blue staining. So what do you do under these circumstances?. Well this apparent failure, led us to see if we could concentrate the protein using ammonium sulphate and acid precipitation. As you can see left. this did do the trick (to some extent, and ideally, we should have dialysed the samples before loading, to neutralise the pH and remove the salt), and indeed confirmed a lower than expected protein concentration in the extract the supernatant, as before. We still see a major band at high molecular weight and little in the body of the gel.

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I was bothered that the larvae contained a major protein and little else and I also wondered why the supernatant was so viscous. The lipid was largely removed, but I wondered about nucleic acids: DNA and RNA. So Michael ran an agarose gel and stained for nucleic acid with ethidium bromide. The first impression is that there is high molecular weight DNA (top of the gel) and a smear of RNA (which at this stage I expect might be rRNA, but this needs further work). Since our starting material was freeze dried,  the high molecular weight of the DNA is a bonus (it is indicative of minimal degradation), but the RNA looks to be degraded. The salt in the samples is probably the reason for the "half smile" of the fuzzy RNA bands. Lanes 1, 2 and 3 contain different amounts of sample, nonetheless, it is clear that the supernatant contains significant amounts of nucleic acids as well as protein. This now presents us with an opportunity to use the mealworms for a wide range of experiments in proteomics and genomics.

We have systematically begun to investigate the potential of the mealworm, Tenebrio molitor as our model organism. I believe the results are very encouraging, but I will now look at similar experiments in Drosophila and mosquito for comparisons and methods. The most important aspect of these experiments, is the approach to research in which an experiment is carried out, the results evaluated and experience and knowledge brought to bear on the outcome. The follow up experiments we carried out begin to bring insight into the investigation, but the following questions remain. Why is the proteome so limited? Do we have levels of degradation as a result of using lyophilised material? What is the origin of the brown colouration? The spectroscopy measurements may give us a clue. These are the questions we shall investigate in the coming weeks. 

The message from this is that we failed to obtain high yields of a diversity of proteins from the larvae, but it may be that our methodology is inefficient or our source material is degraded (or prone to degradation through the day). It is also possible that the protein content of the larvae is limited and the high molecular weight band is a storage protein with some developmental role. The unexpected outcome is that the larvae are a good source of genomic DNA, which opens the door to Molecular Biology based studies. This is an example of serendipity, which I shall discuss in due course.

Wednesday, 5 February 2014

What's in a genome?

I was discussing the difficulty in using digital sequence data to distinguish one mammal from another in the lab classes last week. The power of the BLAST algorithm is that you can look at individual genes and see how similar or different they are across wide ranges of sequenced genomes. In fact we often use BLASTP (where P stands for protein), in order to compensate for codon usage differences, rather than BLASTN (where N stands for nucleotide, or nucleic acid). If we BLAST any histone sequences, we immediately confirm the close similarities between eukaryotes, or we say that these proteins are highly conserved in Nature. We can also see patterns of conservation distributed evenly throughout a sequence in some cases; examples of almost identity followed by a region of almost a total mismatch are also found in some searches. These patterns of conservation and divergence provide clues to gene and protein function and the shared functions of genes and proteins that form a key part of the story of Evolution.

However, if Darwin had only used Bioinformatics, rather than observation of living and preserved organisms, maybe it would have taken longer to deduce his theory of Natural Selection in order to propose an explanation for the Origin of the Species? And maybe George Orwell had a good point when he wrote in Animal Farm: "The creatures outside looked from pig to man, and from man to pig, and from pig to man again; but already it was impossible to say which was which"
It is the combination of experimental observation and in silico analysis that brings us the greatest insights into the relationships between living organisms. The similarities and differences observed in a BLAST search, should encourage us to think of the significance in Organismal, physiological, behavioural and molecular terms. After all as Scientists, trying make sense of the complexity of Life and the Universe, we need all the help we can get!

This brings me to Synthetic Biology. If we are to harness Biology in a systematic way, we must use the rational methods of Synthetic Biology (where we combine Engineering standards and protocols with recombinant DNA methodology) in order to test ideas and hypotheses not only to create new opportunities for improvements to to our Health, our economy and to improve the quality of our lives in general, but also to expose our ignorance of Biology, so that we can establish the truth in Nature.

Tuesday, 4 February 2014

Monkey Business: what does it all mean?

I am sure many of you read or heard about the new technological breakthrough that enables scientists to create apes with specific genetic modifications. As the Guardian put it:

         Genetically modified monkeys created with cut-and-paste DNA

So what is "cut and paste DNA" and how does this differ from the experiments to clone Dolly the sheep? The phenomenon that has been harnessed for modifying monkeys is called CRISPR which stands for Clustered Regularly Interspaced Short Palindromic Repeats. So what does this mean? It is a genetically programmed system found in most bacteria that allows specific DNA sequences (genes) to be inserted at a specific location in a chromosome, this means that a gene for a particular disease can be engineered into an organism that is very similar to humans. This allows us to test drugs in a better controlled setting. There will be those who consider this a dangerous step and those who see it as a major breakthrough. We looked at the history of model organisms in an earlier blog, so how does this development compare? What are your own views?
The problem with the use of model organisms or cultured human cells in the evaluation of new drugs lies in the differences between species and not the similarities. While there may be some drugs that target functions that are common to mice and men, there are some diseases that are organism specific. This is particularly true for pathologies associated with the nervous system and in particular the brain. Clearly, any unnecessary use of animals is to be avoided, but if we are to rely on data from animal experiments, we must be sure that we do not repeat the mistakes of the past: recall that thalidomide was successfully tested on rats and rabbits and given the all-clear. Any advances in developing a deeper and robust understanding of human pharmacology is to be welcomed.

How did Dolly the sheep get cloned. In Dolly's case, the nucleus of a set of breast cells grown in culture was carefully removed and the egg cell, reprogrammed with nuclear DNA. Shortly after, it was clear that this approach worked, but did epigenetics cause the cells to lose their DNA: Maybe we may never know? Dolly grew past pregnancy, but ended up with major chromosomal defects, caused in part by a loss of epigenetic signals and abnormal activities of the chromosome maintenance enzyme: telomerase, which prematurely aged Dolly. 

Molecule of the month January 2013

This month's molecule isn't in fact a single molecule, but rather a class of molecules referred to as both antibodies and Immunoglobulins, or Ig for short. In fact Ig molecules come in several categories (or classes): IgG, IgA, IgM, IgE and IgD. However, they share some common structural and functional features. The classic structure of an antibody molecule (IgG) is shown on the left and is often considered a Y shape. The top arms of the Y provide two surfaces for interaction with the antigen, or invading material which can be a cell, a virus, a foreign object, as long as it can be "recognised" by the so called Fab region (which stands for Fragment antigen binding). The Fc region (of Fragment crystalline) is part protein and part carbohydrate (or Glycan) and is the part of the molecule (or moiety) that interacts with blood cells called B cells or B lymphocytes. The B refers to the receptor proteins on the cell surface that bind to the Fc region of an antibody when the antibody is engaged with an antigen.

The immune response is triggered when an antibody recognises an invading antigen. The two arms embrace the antigen and the IgG (typically) then has an increased affinity for the B cell surface receptor (right). A series of reactions ensue and the antigen is eliminated. (This will be the topic of a later Blog). The antibody molecule is an excellent example of structure related to function. The arms recognise the antigen and the Fc region recognises the cells that eliminate the antigen: antibody complex.

But, I hear you ask, don't we need an antibody for every invading antigen? Re we born with a set of antibodies that protect us from influenza,E.coli, asbestos fibres, cholera etc? Well, we are born with a "library" or antibodies, but only a few molecules of each and we acquire antibodies from our mother's milk. But we also have a machinery that allows us to generate more antibodies than we originally inherited. This is a complex area of Biology and I will only give you a taster. First of all, when an antibody binds to an antigen and then to a B cell. the B cell (which is an antibody factory) produces lots of that specific antibody (we call this clonal expansion). This is one important part of vaccination (more in the future!). We are also able to mix and match segments of the genes that encode the antigen recognition component of an antibody and add it to the common Fc region. So like the chap on the left, we have one head and body (Fc) and a whole set of different hats (each one having a different appearance and recognising different antigens.

What about IgM, Ig E and IgA. These are more specialised antibodies and they look a little different. IgE looks like IgG, but is responsible for your allergic response, recognising pollen grains and triggering histamine release from Mast Cells. Ig A is two copies of an IgG (a dimer) that is found in mucous, saliva, tears and milk. Finally, IgM is a monster (see right) it is a pentamer of Igs, which forms rosettes of antigens. The fundamental unit is a little like IgG, but it assembles into this five membered ring. It is thought to be responsible for the early stages in the immune response, arriving on the scene before IgG.

So, what a great set of molecules and of course, at Cambridge and Switzerland, Cesar Milstein and Georges Kohler discovered a way of making specific antibodies in the lab in the early 1980s. These monoclonal antibodies have been harnessed recently to attack cancer cells. They are the biggest selling therapeutic molecules in the world today. Ever since Rodney Porter (at Oxford, left) and Gerry Edelman in New York worked out the building plan of antibodies in the late 1960s, these molecules have gone from interesting Biology, to useful lab reagents to exciting new therapeutic agents. I hope this has whetted your appetite to find out more!