Monday, 31 March 2014

What's green, glows in the dark and is found in jellyfish?

The molecule for this month is GFP, the green fluorescent protein from the jellyfish Aequorea victoria. This protein has generated considerable interest in experimental biology, and its applications in cell biology in particular warrant its inclusion as a molecule of the month. It is a naturally occurring protein with a function that remains controversial in its normal host organism. However, by combining the power of fluorescence detection with gene fusion and cell transfection technologies, the GFP has made the experimental investigation of cellular processes much more illuminating!

So how does it work? First we need to understand fluorescence. If you look at the definitions of fluorescence, they require you to understand luminescence. So first, luminescence is the emission of light without the input of heat. This is in contrast to incandescence, which is the emission of light that arises when you heat a metal object, such as a light filament. The energy input in the case of fluorescence is electromagnetic radiation: the absorption of a photon of light leads to excitation which is followed soon after (usually in a few thousandths of a second or less) by the emission (or release) of light at a longer wavelength (remember the longer the wavelength, the lower the energy). For completeness there are two other phenomena that are similar. Phosphorescence is very similar to fluorescence, but instead of an almost instantaneous emission of light, there is a delayed release, which is a consequence of a different pathway of excitation and relaxation of electrons in the molecule. Chemiluminescence is the emission of light during a chemical reaction (this is important in some forms of DNA analysis and in the detection of antigens using some antibodies).

The beauty of GFP, is that the physico-chemical origin of its fluorescence does not require the addition of a co-factor: it is entirely embodied in the primary structure of the molecule. Hence, by introducing the gene encoding GFP into any cell, there is no metabolic barrier to eliciting a fluorescent signal. There are of course other reasons why a recombinant GFP might not express its fluorescence, including failure to express (as a gene, or as a translation product), degradation in the host cell by proteases. It is also possible that the expression of GFP is not coordinated in time or space with the process being investigated. But, most of these hurdles can be overcome by systematic investigation. Professor Roger Tsien (who shared the Nobel Prize for his work on GFP), published a subtle mutational variant of GFP in which a serine residue was changed to a threonine. This led to a significant improvement of the GFP in respect of its spectral properties: this mutant forms the basis of most applications of GFP in molecular cell biology. You should think carefully about this mutation by looking at the structures and considering the fact that in contrast to GFP, these two residues are "viewed" as essentially the same by many protein kinases.

Perhaps the most powerful application of GFP is the the revolution GFP has brought about in biological microscopy. Pre-GFP, it was extremely difficult to identify molecules under the light microscope. The methodology often required combining antibody recognition, with the use of small fluorophores such as fluorescein. However, this methodology is specialized, relatively expensive and time consuming. By coupling GFP to a target molecule (using relatively simple cloning technologies), it is now routine to identify a protein molecule and observe its behaviour in the cell. This can be useful in the study of drug targets, fundamental understanding of the role of a protein in time and space and in identifying the way it interacts with other proteins. It is in fact possible to "tune" the fluorescent properties of GFP making it yellow or red! The advantage of this, lies in the ability to measure protein proximity, by the fluorescence changes that can often be observed when two fluorophores come together. This phenomenon is called fluorescence resonance energy transfer, or FRET.

Scientists at MIT, use GFP to monitor 
cell death in their search for new drugs
GFP is a classic "reporter" of events in a Biological experiment. That is a molecule providing indirect evidence for the activity of another protein or gene, a cell or even an entire organism. For example by introducing the GFP gene on a plasmid molecule, that may contain another gene of interest, we can be confident that the cell has taken up the DNA. If we insert the GFP gene downstream of a gene that is switched on in the presence of a hormone for example, then we can investigate that gene expression process and even target it for drug development. We can couple GFP to genes that are thought to play a role in the development of an organism and monitor the timing and location of expression. GFP has truly transformed Molecular and Cellular Biology and its impact. You can see the images used by Professor Tsien in his Nobel address here.

Saturday, 22 March 2014

Science is global, so how do the nations compare?

Science is a global activity, it always has been, but as with most competitive activities, if we take a historical snapshot, there will be one or two countries that make the greatest contributions. If we look at Science from its origins, then the emergence of the foundations of mathematics, physics, chemistry, biology and medicine can be traced back to the Middle East, China, Greece and India. The early developments in Science where largely driven by a human desire to understand the motions of planets and the Natural world together with our own bodies. Moreover, the developments in mathematics by the Greeks and Babylonians; Euclid, Archimedes, Pythagorus and Kiddinu, had enormous implications for engineering, navigation and architecture. 

The next most significant period was probably the age of enlightenment during which time Galileo and Newton (amongst many others) developed a platform for modern, experimental science, founded on robust mathematics. From a biological perspective, I think most would agree that the development of the concept of evolutionary theory, alongside an appreciation of geological time scales set the stage and focus for our current experimental investigation of biological phenomena. I would encourage you to look at the many interesting internet resources (start with a search for the history of science and wikipedia). As a student, I was particularly taken with the 1954 book, Science in History by the eminent X ray crystallographer JD Bernal (pictured right), which provides a thoughtful and stimulating survey of Science through civilisation. I also recommend Jared Diamond's more recent book, Guns Germs and Steel as a scholarly study in why some humans seem to have acquired more "stuff" (or cargo, as he puts it!) than others. I shall post some books that you may wish to read in subsequent Blogs.

If we look at modern science, let's say the last 100 years, it has been a period in which time Newtonian physics has been overshadowed by quantum physics and molecular biology is beginning to challenge chemistry as the Science underpinning manufacturing; not forgetting the Turing-led computer revolution! So the first half of the last century was Euro-centric, whilst the second half was dominated by the USA. It is clear that cutting edge science is becoming increasingly global and is closely linked to economic power. So, whilst stem cell science surfaced in laboratories in the USA and Europe, the Chinese and Koreans are investing massively in these areas (see the 5 circles of patent hot-spots, top left). And those nations who manage to translate their investment in these contemporary scientific breakthroughs into wealth creation and sustainable improvements in the quality of life of their citizens, will surely be the dominant political forces in years to come.

I am going to take a look at how different nations and groups throughout the world conduct science through interviews and exchanges with professional scientists. For example, Professor Vladimir Kramarov and his longstanding colleague Dr. Konstantin Ignatov are two Russian scientists whose research on the development of novel application of enzymes in molecular biology, has been carried out under very challenging times in Moscow. They have agreed to give us an insight into Science in Russia. I will be talking first with Dr. Antal Kiss, from the Biological Research Centre of the Hungarian Academy of Sciences in Szeged (group photo left, Antal is on the right), whose group works on synthetic and systems biology, with a focus on engineering nucleic acid modifying enzymes. Despite the difficulties in obtaining laboratory funds in Eastern Europe and the former Soviet Union, the quality of the science in these two laboratories is remarkable, largely as a result of their commitment and passion for Science.  It is also a consequence of the great traditions of Science in Russia and Hungary and it is important to understand how different nationalities and cultures have contributed and continue to contribute to the body of scientific knowledge. I shall shortly give you an insight into work from these parts of the world through my UTC Blog site.

Monday, 17 March 2014

Y12s welcome Professor Phil Ingham to the Innovation Labs

During Thursday morning this week (20th), I am delighted to announce that we have Professor Phil Ingham visiting the UTC Innovation Labs. Take a look at his guest Blog, from a few weeks ago. Phil will be meeting staff and Y12 students during the lab class on Thursday morning. 

Phil has made major discoveries in the field of developmental biology, using genetic methods and model organisms ranging from Drosophila to Zebra Fish, to dissect the molecular and cellular pathways that underpin the fundamental steps in development. 

Let me know if you want to meet him, otherwise I will show him round the lab as we begin to extract genomic DNA from Synechocystis, and test its suitability for PCR with primers targeted to a specific set of photosynthetic genes.

How to organise and package a genome suplement!

The Y10 class have been sharpening their lab skills by using restriction enzymes to cut the genomic DNA of bacteriophage Lambda and analyse the sizes of the DNA fragments produced, using agarose gel electrophoresis. We refer to this as restriction mapping and it widely used in Molecular Biology to analyse genes, clone genes and to compare regions of two or more plasmids and viral genomes. The skills required include experimental planning, careful pipetting of one thousandth of a ml samples, and the loading of these samples onto agarose gels. I have reported on progress earlier. In the forthcoming class we are going to look at the analysis of data in detail. Not only shall we determine fragment sizes on gels, we shall also look for patterns in the data, as we assemble the restriction digests into a linear order. In addition we shall use the formulae developed by Archimedes over 2000 years ago, to calculate the volume of a genome and the theoretical space occupied by viruses in their host. 

Assembling the lambda genome from restriction mapping data can be frustrating, owing to the difficulties in sizing DNA fragments on agarose gels. The problem is that whilst short DNA fragments do migrate at a faster rate than larger fragments, the relationship breaks down over the range of fragment sizes we wish to analyse (see left). You can plot the distance migrated by a group of fragment of DNA on a gel and you will notice the relationship is non-linear. Simply, small, adjacent fragments appear to be better separated than large adjacent fragments (look at the fragments 1371 and 1264, compared with the bands at the top of the gel). As the size of the DNA increases, the separation achieved by conventional electrophoresis becomes less effective. For this reason, size standards that match the DNA fragment size under investigation are used. Alternatively, in order to overcome this problem, the use of sophisticated power supplies, which pulse the electrical field may be used.

The lambda genome contains just under 50 000 base pairs and since DNA can be approximated to a cylinder, it is therefore possible to calculate the volume of the genome (let's forget the charged surface for now and assume it is a simple cylinder) using Watson and Crick values for the diameter of the duplex and the number of base pairs in a repeating unit. We also know from the pioneering electron microscopy work in the 1950s, the dimensions of the phage head, or capsid and the volume of a typical cell (let's assume both can be represented by regular spheres of 60 and 1000nm respectively). So if the diameter of a DNA duplex is 2nm, 10 base pairs repeat every 3.4nm, we only need the equation for the volume of a cylinder (and it is still pi week!). We might then calculate the volume of the capsid and cell and think about how the DNA is packaged and the maximum number of phage particles that can be squeezed into a bacterial cell (for this you will need the formula for the volume of a sphere). 

1  2   3  m
The first thing I realised is that the restriction digests are still proving to be a little demanding (compare Lanes 1 (perfect), 2 (partial) and 3 (uncut) across the whole class, with some notable successes (see left) and some problems still persisting. The partial is a result of insufficient enzyme, incomplete incubation or inaccurate addition of water/buffer. The uncut DNA is more worrying, but I suspect resulted from a failure to add the enzyme. The lesson here is to take more care over assembling reaction mixtures and if you are unsure about using any equipment, please ask first! On the positive side, we are getting nice gels and the experiment is probably working for 50% of the groups. Take a look at your lanes on the class data for today (Innovation Portal) and make sure you note whether your sample gave the predicted pattern and comment on whether the reaction went to completion of not.

I was pleased to see that the maths part of the session went so well. I realised that graph plotting and the use of logs and log graph paper is completely new to you, but the number of students who elected to use semi log paper was very satisfying. I think we are starting to see evidence emerging of the value in thinking about the best way to plot data and your ability to observe trends in data (here it was the non-linear migration of DNA fragments in agarose gels). The 2 cycle semi log paper might be improved on if we use 2 cycle on both axes next week. I also decided to stretch you on the volumetric calculations: determining the volume of the cylinder that best describes the linear lambda genome of 48,500 bp. You solved this pretty quickly and I could see that you then found the calculations of the volumes of phage capsids and bacterial cells (assuming they are regular spheres) was then straight forward. In the end, I felt that we had come away with an appreciation that some numerical relationships in Biology are best described in non-linear terms and that there is significant value in being able to calculate lengths and volumes in order to firm up your understanding of size relationships as we consider the molecular and cellular basis of phenomena phage and viral infection mechanisms.

Friday, 14 March 2014

First week of the Synthetic Biology Project: PCR success!

It was a foggy on Thursday morning when I walked into the Innovation labs and Michael was distributing the reagents, template, primers, dNTPs etc. The Y12 group were due to arrive in an hour and by the end of the afternoon, all of their skills training would be put to the test. I also had to fit in an explanation of the fundamentals of DNA replication: the enzymes, the concept of primers and the polarity of DNA synthesis, not forgetting the logic of the thermal cycling process. By 10am the student teams had assembled all of the reactions and the new PCR machines were cycling (only for the second time!). By 1.30pm the gels had run and the results were in! Out of the 16 groups we had two failures, which I think is pretty impressive and one of the gels is shown on the left. No need for labels, the PCR product is clear in all but one reaction mixture. This means that we have now worked through all but one of the Y12 target methods, (ligation and transformation the only experimental method that we have yet to complete before Easter vacation). With these methods in hand we can now isolate the gene encoding non-ribosomal protein synthesising machinery from our Blue-green algae. Moreover, Y13 is set to become a great opportunity to exploit these skills and pursue a range of customised projects.

But back to Thursday. The Polymerase Chain Reaction (PCR) results in the sequential amplification of DNA through the polymerisation of new DNA strands, driven by a heat stable (thermostable) DNA Polymerase. Two strands of the template duplex are denatured and form the templates for primers and polymerase to generate copies. Two strands become, 4, 4 become 8, 8 become 16 and so forth until millions of copies are produced in a matter of hours. Not only does this facilitate DNA analysis, it is the core of many diagnostics and forensic methods. In short, it is possibly the most widely used method in Life Sciences today. But it also provides an opportunity to appreciate mathematical relationships.

DNA amplifies in an exponential manner, but the use of logs and their value in plotting data that span large experimental ranges, is not covered by the National Curriculum, unless you take Maths A level. No surprises then that understanding pH, or estimating binding affinities and physical relationships in Biology, essential in many diagnostic and analytical procedures, proves so challenging for first year undergraduates  and new lab technicians. So I am delighted to say that we used our time in between amplification and electrophoresis, to explore the value of logarithmic plotting methods (a picture of school log tables is shown left, for those of you over 50!). We plotted fragment migration on gels, we have previously looked at bacterial growth curves and what was really encouraging was the enthusiasm the students expressed for "having a go" at something that is often perceived to be too challenging. So, not only did we get the technique of end point PCR nailed, we have paved the way for understanding real time PCR analysis and more generally, non-linear experimental data, which pervade Biology. 

Friday, 7 March 2014

You've got to admit, we're getting better....

Those of you who have to suffer the annoying comments from your granddad, or your mum about how pop music isn't what it used to be since the Beatles split up, might recognise the title of my Blog today (nearly, from Sgt. Pepper's Lonely Hearts Club Band). What's my point? The Y10 class carried out the second day of their lambda genome mapping project last Tuesday. On the first day, I inserted a skills test to assess each student's ability to set up and analyse a restriction enzyme digestion of lambda DNA. The results are compared below. This strategy has been introduced into the projects to measure the development of your skills. The ability to deliver micro-litre volumes of nucleic acids and restriction enzymes, followed by loading onto agarose gels, might be something professional laboratory scientists do every day, but isn't common among 14 year olds!

Week 1 (left)
Week 2 (right)

So how did yo do? Above, on the left is an example of a gel in which you digested lambda DNA using the enzyme EcoRI (in week 1): on the right is the gel analysis of the same experiment carried out a week later. I don't think I need to say anything more about the level of improvement! Congratulations to all students in the class, this is an example of how data of a "publishable" standard can be achieved in the Liverpool Life Science UTC labs with dedicated students honing their skills through our Innovation programme: at the age of 14!

The images on the right are taken from open access publications and show the quality of the class data above (week 2) compare favourably with professional science labs. The first (left) is from a well respected journal in the Tropical disease field and the second (right) is from a University undergraduate team entering an international molecular biology competition. I think you will agree, we are on our way!

Why should I go to a seminar when I could be....

Research seminars are one of the most powerful means of presenting and discussing scientific data. They have been around for many years and as a conservative estimate I think I will have been to over 3000 since I started my career. Some have been given by undergraduate students, many by PhD students and the majority by academic visitors to the various Institution where I have worked, in Europe and the USA. At best a seminar can provide insight and inspiration, at worst a seminar can be shambolic, delivered in a rambling, disorganised manner based on poor quality data. Maybe it surprises you but both ends of the spectrum can provide equally valuable experiences. However, what is more important at this stage, is that everyone participates in the UTC seminar programmes, whether it is attendance at a Master Class, presenting your own project work, listening to your colleagues and most importantly, actively participating in the question and answer sessions.

We had the presentations on Thursday from the Unilever project, in which you were all asked to investigate the antibacterial activities of plant extracts of your choice. The project introduced you to methods of screening bacteria, methods of extracting water and ethanol soluble compounds from biological material ; it also incorporated a wide range of transferable lab skills and generic skills, including time management, sample labelling and storage (as discussed in an earlier blog). The presentations were all excellent and we (me and the teachers who were present) selected the Perutz and Hubble teams to go forward to present to the industry sponsors. It may sometimes seem unfair when presentations and posters are judged in this way, but that's how it is in the outside world! The successful award of research funding and the acceptance of published work in scientific journals is highly competitive and is typically judged by the process of "peer review". One of my jobs is to make sure that you leave the UTC fully aware of these issues and "match fit" for a career in the Life Sciences sector.

What made those two presentations the winners? The
overall quality of the presentation was an important factor, clear information and images on each slide, rather than large amounts of difficult-to-read text. We looked for imaginative ways to convey the significance of your results and the way in which methods and background research were explained. The final (and for me most important) criterion was data analysis and presentation. Your use of graphical methods to extract the significance of data and to quantify outcomes was a key aspect in the selection of the winners, but importantly compared with the last presentation session, I saw major improvements across the class, in your understanding of the Science that we are exploring.

How can you improve on the presentations? Apart from the issues of clarity of explanation on the slides, one simple way of improving is to practice! The use of unfamiliar technical terms can be overcome by asking me how to pronounce it, or alternatively listening to it being spoken through an online dictionary. The other thing to avoid is reading out text and facts etc that you do not understand. I listened to some nice explanations of some difficult concepts, but when I pushed you in the Q & A sessions, on occasions some of you were reading out lines with little understanding. I know that you are being stretched, but try and keep to what you know and anything you are unsure about explaining leave out.....for now!

Why is it not only important to give presentations but also important to attend those of others? First, we can all learn from others, at all stages in life. I have learned huge amounts from talking to students, staff and colleagues at all levels in my career and hope I continue to do so.  [I have really developed my understanding of molecular spectroscopy by having to prepare classes and field your questions on the properties of organic dyes.] A seminar may have an interesting presentation style, and may make use of novel ways to explain challenging phenomena. On the other and you can see how some things don't work and you can avoid them in your own talks. By attending seminars and presentations in the lab, you are not only being respectful to your colleagues, you are also giving your support! If you want them to listen to you and engage in questions (the added value of seminars), you should respect them too and come along to their presentations. Constructive criticism at the UTC in a supportive culture is, in my own view, one of the best ways to prepare you for your working life. In short, if you want to be a well rounded scientist, you must embrace the seminar culture wholeheartedly. 

Finally, for all of the above reasons, your commitment and performance at the UTC will include an assessment of your engagement in the seminar programme. 

Tuesday, 4 March 2014

Guest Blogger Professor Phil Ingham FRS

I am delighted to announce that a number of Internationally renowned Scientists have agreed to post a series of Blogs for the UTC.  The first is from Professor Phil Ingham FRS, a geneticist who was born and educated in Waterloo, Liverpool (and is still a keen LFC fan!) but is currently based in Singapore where he is the Toh Kian Chui Distinguished Professor of Developmental Biology and also the Vice Dean for Research at Lee Kong Chian School of Medicine, a partnership between Imperial College, London and Nanyang Technological University, Singapore.

Phil's work on the fundamental pathways that control animal development has been recognized by numerous awards, including the Genetics Society Medal in 2005 and election as a Fellow of the Royal Society in 2002. You may recall in an earlier Blog I mentioned the hedgehog gene. This was discussed originally on the Cancer Research UK site, by their Science Communications team. I will also be posting a follow up interview with Phil later in the year, similar to the one with Rich Roberts.  Here, Phil begins with the background to his work that led to the discovery of an exciting new cancer therapy: it started with fruit flies and zebra fish, model organisms that have been pivotal in identifying the genes that control the complex processes of animal development, many of which when mutated can cause cancer.

"Curiosity in basic biological phenomena with an eye for their relevance to human disease can best define my approach to research over the past 30 years. Genetics is a tremendously powerful discipline because it gives us an entry point to the molecular mechanisms underlying biological processes without any prior knowledge of their molecular basis. The identification of mutations affecting cell division in yeast or the differentiation of the appendages of the fruit-fly, Drosophila, for instance, has provided us with major insights into cell cycle control and the control of embryonic development respectively. As more challenging questions emerge in the Life Sciences, we need to consider increasingly complex model organisms, to investigate the genetic basis of our own physiology.

For this reason, my lab turned to the zebrafish in the early 1990s and cloned the homologue of the Drosophila hedgehog gene from this organism. Hedgehog (Hh) proteins belong to one of the handful of families of signalling molecules that regulate animal development. Dysfunction of the Hh signalling pathway results in severe developmental defects and is associated with a number of different types of tumours in man. Although the pathway is highly conserved through evolution, there are some important differences, particularly between Drosophila (the species in which most is know about the mechanism of Hh signalling), and vertebrates. In my lab, we have used a combination of genetic and proteomic approaches in fruit flies and zebra fish to explore both the conservation and divergence of Hh pathway mechanisms and function. 

These studies paved the way for a drug discovery programme initiated by  a small biotech company in Cambridge Mass in the late 1990s and culminating in FDA approval for the anti-Hedgehog drug  Vismodegib in January, 2012.Vismodegib (pronounced vis-mod-ee-geeb and shown left) also known by its brand name Erivedge (and unhelpfully by the number GDC-0449!) is one of a growing number of “smart” drugs, designed to target specific biochemical pathways that underlie different cancers, in this case, metastatic basal cell carcinomas. These are tumours that start in the skin but spread to another part of the body and are very difficult to remove by surgery. A common feature of these tumours is that they over activate the pathway that is normally controlled by Hedeghog proteins.  Genentech, a pharmaceutical company that has pioneered the development of drugs that target complex biochemical pathways, developed Vismodegib in collaboration with the biotech company Curis Inc.

Another import aspect of my career has been the mentoring of research students and early career scientists (postdocs, or post-doctoral research assistants and fellows, to give them their full title). A large number of both categories of researchers have passed through my own lab over the years, many of whom now occupy very senior positions, for instance as full Professors or Directors of research institutes in countries around the world. 

One of the most challenging but rewarding experiences, was the opportunity to develop a cluster of Developmental Geneticists at the University of Sheffield, an initiative that was eventually awarded Centre status by the Medical Research Council. After moving to Singapore, initially on secondment from the University of Sheffield, I became Deputy Director of the Institute of Molecular and Cell Biology, the oldest biological research institute in Singapore. It was very interesting and exciting experience helping to run an institute in such a vibrant and fast moving country as Singapore, and the experience has set me up well for my latest appointment as Vice Dean of the new Lee Kong Chian School of Medicine (left) where I have full responsibility for the development of its medical research strategy. These are exciting times in medical research with immense problems in health care facing us but enormous opportunities to address them through the application of emerging technologies and the explosion of biological knowledge that has happened over the past three decades. Hopefully, many of you reading this blog will contribute to this effort in the coming years!”

Sunday, 2 March 2014

Genome mapping with Restriction Enzymes: Y10 Lambda Project Part II

The use of Restriction Enzymes (or to give them their proper name, Restriction Endonucleases) to selectively cut the genetic material, DNA, opened the way for screening patients for genetic diseases around 30 years ago. These enzymes are isolated from bacteria and naturally recognise and cut specific, short (4-8 base-pair), sequences of DNA. We have been generously supplied with a range of these enzymes by New England Biolabs for our lab project. The most "famous" Restriction Enzyme is probably EcoRI (the name is derived from the organism it is extracted from [E.coli] the strain [R] and the order in which it was discovered [I,II etc]). Hence the restriction enzyme BamHI comes from Bacillus amyloliquifaciens, strain H, and it was the first such enzyme to be isolated from this strain.

In the 1980s in particular, the combination of these enzymes and the use of radioactive phosphorus to detect minute quantities of DNA, made it possible to detect differences in the properties of genes in patients suspected of passing on genetic diseases during pregnancy. The technique for detecting the DNA fragments generated by these enzymes was known as Southern Blotting, after its inventor Professor Ed Southern (on the right there is an image of one such diagnostic blot). Under some circumstances, genetic mutations would give rise to changes in the restriction enzyme patterns, which would help doctors diagnose genetic diseases as a first step towards treatment.

Phage lambda DNA is a linear duplex of DNA, when isolated from the phage particles. We will use this DNA as a substrate for a set of restriction enzymes. The enzymes will be incubated with the DNA and the cleaved fragments run on an agarose gel. The banding patterns will be recorded and the sizes of the DNA fragments measured alongside some DNA fragments of known size (usually expressed in base pairs). The lambda genome can be analysed using free online software in order to determine the position of our restriction sites and the genes that control the development of the phage. In this way we shall combine experimental analysis with the use of Bioinformatics, just as Molecular Biologists do in their research work.

Saturday, 1 March 2014

Understanding genomes through bacterial viruses: the Lambda Project for Y10. Part I

E.coli under attack from
phage lambda
The viruses that infect bacteria (or prokaryotes) are called bacteriophage (a combination of the Greek words bakteria, a staff or cane, in view of the common rod-like appearance of many bacteria under the microscope, and phagein, which means to devour). They are the most abundant organisms (see below) on our planet and are often simply called phage, for short. As with all viruses, they cannot replicate without "help" from the host they infect. You will be more familiar with the influenza (or flu) or the cold viruses, which are viruses that infect us. However, we stop short of referring to viruses and phage as true life forms, since they require help from the host cells that they infect. The image left shows the phage lambda being infected. Sometimes the phage burst open the cells with the release of thousands of phage particles (we call this the lytic phase). Alternatively the phage infect and then adopt a dormant phase, where they just sit and wait before some external event triggers the lytic phase. Bacteria carrying such phage are called lysogens. The switch between these two phases is controlled by a genetic circuit. This is a simple model for the decision processes that take place in us, although the system is much simpler. We are going to use a set of "molecular scissors" to investigate the genes that make up phage lambda.

Because viruses and phage require host cells to replicate (copy themselves), they are usually simple particles, with their genes (in the form of DNA or RNA) packaged in a shell, often made up of a small number of proteins. In the case of phage lambda (left), around 30-40 genes encode the same number of proteins requiring a total of just under 50 000 base pairs of DNA. The phage head and tail (right) are encoded by the phage. When the phage infects and "sleeps" it enters the genome of the host. When it is awakened, it leaves the host genome and sometimes takes some of the host genetic material with it. This phenomenon is known to occur in animal viruses too and was critical in the isolation of cancer genes, where certain cancers are transmitted by viruses.

Phage lambda is encoded by just under 50 000 base pairs of DNA in a linear chromosome. Last week you used an enzyme called EcoRI to cut the DNA into pieces and then used agarose gel electrophoresis to "visualise" this. Last week's experiment was aimed at ensuring you could carry out the manipulations (microlitre volumes etc) sufficiently accurately for us to carry out the detailed mapping that you can see left. I have posted your data on the Innovation Lab Portal. We have some work to do to get the gels up to scratch, but we will have last week's experience behind us. I shall explain the use of these "molecular scissors", or restriction enzymes in Part II. They will help us map the genes on the phage, with the help of the lambda genome sequence that we can easily access through NCBI.

Penicillin: molecule for the month of March

Penicillin is one of the most well known of biological molecules, although I suspect that most people who know the name, don't know the molecule! The structure of penicillin shown left. The compound comes in a number of slight variations which are generated by changing the chemistry at the R position (top left). (The use of the letter R is common in chemistry and biochemistry for indication a range of functional groups). The effect of the drug was famously discovered by Sir Alexander Fleming in 1928 (for which he later shared the Nobel Prize with Florey and Chain). The square shaped "ring" structure at the core of the molecule is called a beta lactam, which is fused to a five-membered thiazolidine ring, which gives rise to the term penam, and hence the name penicillin. 

The compound was first extracted from the mould, Penicillium, thanks to the tenacity mainly of the Biochemist Ernst Chain (it would be Florey who pioneered its application for the treatment of clinical infections, although my colleague at Sheffield, Milton Wainwright has I believe, produced the definitive Penicillin history). It acts on the enzymes responsible for the completion of the biosynthesis of the bacterial cell wall. The polymers that give the stiffness to bacterial cell walls are a mixture of carbohydrate and amino acids and are called peptidoglycans. The drug prevents these polymers from forming cross-links, which give the bacteria an outer netting. 

Penicillin looks unusual as most biological molecules go and it is in fact synthesised by an interesting class of enzymes that form the basis of the Y12 synthetic biology project. The compound is formed by joining three amino acids together Amino Adipic Acid (not an amino acid found in proteins) and the two proteogenic (protein generating) amino acids Cysteine and Valine. The enzyme name is ACV synthetase. It is one of a group of enzymes that assembles amino acids into short peptides, giving rise to the term non-ribosomal peptide synthesis (since most proteins are made by the ribosome). The molecule is then further modified by a group of enzymes, to produce slightly different forms (the image left is one such enzyme). 

Penicillin remains a major drug for treating infections, and is often used in conjunction with other antibiotics if infections are persistent. Except for the 1% who are allergic to it! The problem of resistance to penicillin is also a major problem. Here the target for the drug has picked up a mutation, and is no longer inhibited, allowing cell wall biosynthesis to continue unhindered. The careful and responsible use of antibiotics is a hot topic at the moment, and the search for new ones, should, in my view be a greater priority.