Saturday, 31 May 2014

Twenty skills for a lab technician in 2014

Experimental science, whether it is analytical (diagnostics and forensics for example) or discovery based (basic or applied), requires the "mastery" of a set of practical skills. But what skills? When I look at the lab skills that form part of the schools' curricula in chemistry and biology, I see a major mismatch with those in my own research lab at the University of Sheffield. There are undoubtedly fundamental skills in many of the common experiments: titrations, dissections, separations etc. All of which I too benefited from as a scholar in the '70s. However, charged with the preparation of UTC students for University research labs and the work place before the first cohort leave in 2015, I thought I would define the 20 most important skills. I am now seeking opinions and alternative suggestions from our partners, but would like to widen the net further and this "Micro-Blog" is aimed at getting the ball rolling. Before I make my final list, the order isn't meant to signify importance, although this might emerge through dialogue!

1. Using and calibrating a pH meter
2. Using and respecting the cleanliness of a balance (top pan and fine balances)
3. The ability to make a solution at a given molarity (and recognising the appropriate apparatus to use)
4. Using and maintaining a "Gilson" (or equivalent)
5. Safe use of a centrifuge (from bench top to floor standing)
6. Sterile technique for microbial growth on plates and broths
7. Preparation of nucleic acids (genomic DNA, plasmids and RNA)
8. Microbial cell disruption for protein preparation
9. Agarose gel electrophoresis
11. Column chromatography (ion exchange, affinity and possible gel filtration)
12. Keeping a legible lab notebook
13. Ability to produce a daily work plan
14. Understanding the storage requirements for experimental samples
15. Logical and organised storage of reagents and materials
16. Appreciation of visible and UV spectroscopy (others are more specialised)
17. Basic plasmid transformation and mini-prep methodology
18. Designing PCR primers and basic end-point PCR
19. Knowledge of a method of molecular cloning and restriction analysis
20. Use of a light microscope.

What do you think? Any missing, any that should be deleted? I look forward to your responses!

Thursday, 29 May 2014

June's Molecule of the Month: RNA Polymerase, the cell's very own photocopier?

Transfer RNA
We are all (well maybe that's a bit of an exaggeration!) familiar with DNA and genomes, but in the Natural world, RNA is always found, whereas DNA is not! RNA, not DNA is the universal nucleic acid. This is because there are some viruses and bacteriophage that use RNA as the "repository" of their genomic blueprint. Moreover, it is the expression of DNA genomes in the form of RNA transcriptomes that leads to the expression of the phenotype, or characteristics of a cell, and subsequently the organism. Therefore, the central dogma first articulated by Francis Crick: DNA makes RNA makes protein, should be modified to become: "in general, DNA makes RNA makes protein, but sometimes RNA makes DNA, makes RNA makes protein!" The difference between DNA and RNA is subtle. In fact in terms of chemistry, it is simply the presence of an extra oxygen atom on the 2' carbon of the ribose sugar (in DNA, the letter D comes from the deoxy-form of ribose). The presence of this oxygen is sufficient to prevent the formation of a stable B form double helix, without which Watson and Crick would not be household names. It presents a steric barrier to the stable formation of the so called B-form double helix: RNA molecules (like tRNA top left) do often contain double helical regions, but they are usually in short stretches and take the A-form. (Don't worry about As and Bs, they are just historical labels and we also have Z forms which are left handed helices!).

Transcription from DNA 
Many Molecular Biologists would argue that RNA is more important than DNA, but let's just assume both are important. Where does RNA come from? In vivo, DNA and RNA molecules are biopolymers and they are synthesised from a template. That is to say, you need a DNA polymer to make DNA copies in genome replication, and you also need DNA to provide the template for RNA synthesis. (There are some special examples of DNA and RNA synthesis that do not follow this rule, but they wont concern us just yet). Transcription is the name we give to the synthesis of RNA from a DNA template. For example, in order to produce haemoglobin, the organism or cell must first encode the gene (we do, most bacteria have something different). The gene is first prepared for transcription by genome, or more commonly called, chromatin modifying (or remodelling) proteins (imagine unwrapping an orange), the start of transcription often involves a group of protein molecules that mark the start of the gene (the promoter) and these molecules, when in position, provide the impetus for the enzyme (that is the subject of this blog) to bind: RNA polymerase. So technically RNA polymerase is an enzyme that synthesizes RNA from a nucleic acid template using ATP, TTP, UTP and GTP. These NTPs (collective abbreviation) are the building blocks of RNA (recall DNA uses TTP instead of UTP) and their incorporation at the right site is facilitated by the sequence of bases in the template. For example, a DNA sequence (5')GGTTCCAATTTGG(3') would be copied as (5')CCAAAUUGGAACC(3'). (Students, think polarity of synthesis and complementarity of base pairing!)

The enzyme found in E.coli that produces mRNA (and rRNA and tRNA) is simply called RNA Polymerase. In general, one polymerase transcribes all RNA classes in bacteria. In higher organisms, we have an RNA polymerase for each class of RNA: RNA Pol I (rRNA), PolII (mRNA) and PolIII (tRNA). one of the most interesting polymerases is Reverse Transcriptase, which takes RNA templates and converts them to DNA (I will be covering RT as a separate molecule of the month in the future, in view of its special role in AIDS infections).

Eukaryotic RNA Polymerases
comprise multiple subunits

A simple RNA Polymerase will catalyse the polymerisation of RNA, but one of the most interesting features of these molecules (apart from their catalytic mechanism and their level of fidelity), is that they are capable of being regulated in a range of different ways. Organisms from all walks of life are able to activate or repress their RNA Polymerases; or put differently, all organisms can regulate transcription. This may be achieved in one of two generic ways. Firstly, proteins can interact with the core polymerase machinery to change its shape, and as a consequence the catalytic site is either shut down or, in contrast activated. This phenomenon is classically referred to as allostery (from the French for "other site"). In Biology, when an enzyme is modulated in function by the binding of a small molecule, or macromolecule, to a site that is remote from the active site; this is generally a strong indication of regulation. The toggling of enzyme activity in situ, is one of the key mechanisms underpinning the development of complexity in higher organisms (it is also a major "economy saving" device used by cells to control the flux of metabolic intermediates in intermediary metabolism). Allostery goes hand in hand with the second way that RNA Polymerases can be regulated: a phenomenon known as post-translational modification, a topic we shall discuss at a later stage in some detail. However in simple terms, the addition of low molecular weight adduct to a target protein (usually through enzyme-mediated transfer) such as a phosphate or a methyl group, can also induce a shape (conformational) change in the protein, which can indirectly activate or inhibit as with allostery (see the cooperative binding curve for Haemoglobin in an earlier Blog).

Further reading 

Comparisons between the RNA Polymerases in bacteria and animals 

The three eukaryotic RNA polymerases

"On the nature of allosteric transitions: a plausible model": A landmark paper from 1965, in the field of allostery: a substantial paper with a secondary focus on molecular symmetry in proteins. A "must read" for all biochemist. Jacques Monod, Jeffries Wyman and Jean-Pierre Changeux all made major contributions in Science in its widest definition between 1930 and 1990! 

Mark Ptashne: another pioneer in the molecular biology of transcription: from repressors in bacteria to transcription factors in yeast: a remarkable career punctuated by some of the most elegant experiments (he's the accomplished violinist on the left!).Roger Kornberg's lab (awarded the Nobel Prize for determining the structure of RNA Polymerase from yeast): a truly remarkable piece of Biochemistry and X-ray crystallograohy. Robert Tjian: a pioneer in the biochemistry of eukaryotic transcription: I first came across his work in the early '80s along with that of Robert Roeder

Wednesday, 21 May 2014

Embedding 3D Printing in Research Labs

Taken by surprise by the Sheffield University 
alumni team at the Gibson virtual reality suite 
that they helped to fund!

Three 3D printing (3DP) is revolutionising manufacturing. It was during a visit to the University of Sheffield's Advanced Manufacturing Research Centre that Professor Keith Ridgway first made me aware of the power of the concept, which involves the systematic deposition of layers of material, in response to a digital design, to build an object. On the same visit, I was also introduced to the power of virtual reality in working with three dimensional objects from jet engines to protein molecules. A year later my colleagues and I raised funds to install a similar projection system into the Department of Molecular Biology and Biotechnology at Sheffield (top left). The room was subsequently opened by Nobel Laureate Ada Yonath, in honour of the Department's second Head (after Sir Hans Krebs), Professor Quentin Gibson, FRS (1918-2011). The ability to visualise the oxygen binding site of Haemoglobin and watch as the protein's iron centre, attached via a Porphyrin Ring responds in conformational terms provides students and staff with huge insight into Biomolecular interactions, something I am sure Quentin would have loved!

Quentin making, fixing,
but always thinking!
The powerful visualisation of objects in three dimensions is a great way to provide young scientists with a deeper appreciation of the molecular process that underpin life. However, I was recently persuaded by my physics graduate, George Rule, who helps me in the Innovation Labs here at the UTC,to obtain a 3D printer in order to generate molecular models to aid in the teaching of Molecular Biology. Printing DNA duplexes and scale models of bacteriophage particles proved a real success in explaining to the students (ranging from 14-19) the principles of molecular structure and function. However, I remembered my AMRC visit and the impression left, together with something Quentin told me when I visited him shortly before he died in New Hampshire, USA. When I sought his advice on making your way in Biochemistry he gave me the following answer. If you are trying to solve a problem and you are limited only by money for staff and resources, but not by intellect and determination, then you can still be innovative if you build an instrument that measures an important property of a molecule or cell, that nobody else can (that's Quentin top right,  still making things while in his early '90s!). In other words, one way to be a successful scientist, is to incorporate a level of design and manufacturing into your laboratory programme. This is where 3DP comes in.

Mini-racking system to hold 
Pasteur pipette columns
You may have read recently about the publication of a paper that describes the fabrication of a powerful microscope using paper, a small battery and a cheap lens by Professor Manu Prakash . In his work Prakash reminds us that scientific breakthroughs do not always rely on high end, specialised instrumentation (which incidentally usually come in at a high cost in terms of both purchase and maintenance). By embedding 3DP into a contemporary Molecular Biology laboratory, I believe a new generation of scientists will flourish using Quentin's guiding principle. Let me give you some examples of how we are following this concept at the UTC.

My own interest in teaching commercial aspects of Science (Molecules to Market courses) from GCSE to PhD, led me to assemble a group of Y12 students interested in the technical side of Science, to form Greenland Biodesign (an in-house organisation at the UTC, taking its name from the UTC's location on Greenland Street and the students' own idea, since Biotech was taken!).The students have interests ranging from general Biology, Biochemistry and Genetics through to Chemistry, Physics and computer aided design. In between formal classes, the Greenland Biodesign (Web site coming soon!) team are working on individual, basic and applied science projects, but are also driving the organisation of the large scale experiments (100 students) that we carry out weekly in the Innovation Labs. The first UTC project that combines custom manufacturing via 3DP is partly funded by the Royal Society and involves the development of the Mealworm  Beetle (Tenebrio molitor above right) as a "School Friendly" model organism. The aim is to develop Biochemical resources (Proteomics, Genomics and Transcriptomics) and methods for illustrating Biological phenomena from Developmental Biology to the molecular basis of enzyme catalysis.

The idea behind our
Greenland sonicator cup
Greenland ice breaker
Using our standard work flow planning approach (template on the RHS links, if interested) students are asked to work in small teams to develop robust extraction procedures for proteins, DNA and RNA. The first stage of the project is focused on larval biochemistry and utilises meal worms that are bred in house this is a separate programme run by Greenland Biodesign).We also use freeze dried meal worm in some situations (which can be obtained readily from a some supermarkets or on line for small change!). Mechanical extraction (homogenising and grinding) can generate undesirable heat and so we needed to keep the material cold prior to chromatography on our racking system (above left). We use glass homogenisers in some cases (for DNA and RNA), but sonication is something we are planning for protein extraction, since there are some oxygen sensitive proteins released. We have designed sonication cups based on ancient principles (top left!) for sample cooling during extraction  in order to circulate the extracts through ice, during sonication (our designs will all be made available shortly via the Greenland Biodesign web site). 

One of the major challenges that I have encountered in successfully delivering large scale lab projects with very young (14 year old) students (and indeed with some Graduates), is sample management. We use fridges and freezers for sample storage. We are often faced with storing up to 10 samples for 25 different project teams each week; these samples may be needed again during the course of a year. Whilst this is something a PhD student or commercial scientist must deal with every day, it is a skill that we believe needs teaching in a formal manner at the UTC. 

3D printed objects used
in Science education
The Greenland team have designed and fabricated a range of customise tube holders, capable of accommodating Eppendorf sizes to Falcon tubes, Universals, centrifuge tubes etc with mix and match options for different experiments. We have also developed an expandable racking system for the long term storage (and easy access) of samples  at -80. The recent design of a round rack for multiple tube sizes (Greenland Icebreaker, above right), that is free standing without a base, has proved really useful when the sample need to be embedded in ice. 

We are fortunate that we have equipped the lab with simple, but versatile bench top items
Greenland gel formers
such as micro centrifuges (for both simple deposition of microlitre volumes and extract separation), which satisfy lab needs, but we are reaching capacity with electrophoresis equipment and our mini-agarose gel templates and combs (right) are the first step in expanding our DNA separation and analysis capabilities. And all for under a quid per gel former! However, plastic gel equipment in high use by young scientists in waiting, can take some "hammer" and we use 3DP to fix parts, and to replace those components like gel spacers and combs that seem to disappear with the same frequency as socks in a washing machine!

Greenland freezer racks
These are just a few simple examples of how a 3DP facility embedded in a research teaching lab (which forms a key element of our approach to teaching practical laboratory skills and investigative science at the UTC). However, I believe that every University department (or indeed a research group of around 10 students and post-docs) should consider integrating 3DP into their daily lab infrastructure and routine. It is Quentin Gibson's advice that I can hear in my head when I think of the challenges associated with producing competitive Life Science Research (particularly Molecular Biology and Biochemistry) which must be overcome in order to obtain the necessary research funding. By combining creative thinking, robust technical and analytical skills with 3DP, I believe there could be a renaissance in experimental science and I believe the young scientists at the UTC are in the vanguard of this renaissance.

Friday, 16 May 2014

Project Tenebrio: larval proteome emerging from theY10s

This is just an update on our Tenebrio molitor meal-worm project. The last two sessions have focused on developing a robust extraction method for proteins and the application of ion exchange for identifying the most abundant classes of protein. We have decided that the traditional mortar and pestle is inadequate to the task! It proved difficult to obtain reproducibly high concentration extracts across the group, using freeze-dried larvae. We did however make significant progress using the slightly more technologically advanced glass homogeniser (kindly donated by Mel in the Department of Molecular Biology and Biotechnology at the University of Sheffield)! Using this method, and only two larvae (and importantly keeping everything cold on ice) we have been able to obtain extracts in about 30 minutes (following centrifugation) that are suitable for downstream protein (and nucleic acid) purification.

We first asked if the tissue extracts could be resolved cleanly on SDS PAGE and then collected no salt, medium (500mM) salt and high salt (1M) fractions: then Michael ran a range of class samples on a 10% SDS gel. As a result we clearly identified two major species at high molecular weight one eluting at low salt and the other binding tightly at pH7.4 to the Q sepharose (kindly provided by our friends at Eden Biodesign). Dr. Mark Dickman in the Department of Chemical and Biological Engineering at Sheffield has agreed to submit his samples for Mass Spec identification. However I am really keen to identify the low molecular weight protein that eluted a high salt (see below left). It could be one of Tebrio's antifreeze proteins, which are of some considerable biotechnological interest! I will let you all know as soon as we get the results back.

The protein extraction and analysis is running hand in hand with our development of a data base for T. molitor. There are around 6 000 transcripts documented at NCBI and each of the Y10s has been allocated 100 each, giving us complete coverage so far. We are working on a student friendly data set, and we are looking for keen Studio School collaborators to help us develop a user friendly interface or App over the coming weeks. This week we shall concentrate on improving the separation with larger columns and analysis of the nucleic acids. Keep up the nice work and we should soon be able to put together a manuscript for publication from the Y10 Class and Greenland Biodesign. I shall give you an update before half term and the data will be available to students on Edmodo.

Saturday, 10 May 2014

Hungarian Trick!

Hungarian Academy of Sciences
Antal's lab group
I have now had the time to edit Dr Antal Kiss's post to me that gives us a lovely insight into Science in Hungary. I used a Q and A format: I hope you enjoy reading it as much as I have.

Q How did your first become interested in Science and Biology in particular?
A: As far as I can recall, two books had the biggest influence on me.  One of these books, which I read at the age of 12 or 13, was written by György Lányi, a Hungarian biologist and popular science writer. The book had the title: "Life under the surface of water”. Fascinated with the wonders of aquatic life I wanted to become a hydrobiologist. Then,  already at secondary school, I read Paul de Kruif’s famous "Microbe hunters”. After reading this book I decided to become a microbiologist or a scientist working in medical research. 

Dom Square in Szeged with
the National Pantheon
Q Could you give us some insight into the pathway from school to University and to research in Hungary?
A: I think the pathways are similar to those in other European countries. Most students encounter active science when they begin to work on their diploma thesis at a science department, usually during the last two years of their studies. The more ambitious start earlier. The best students present their results at local and nationwide conferences of the  National Scientific Students’ Associations. Presentation at such conferences is usually regarded as an advantage at entrance examinations to PhD Schools.There are institutionalized forms of training high school students who develop an early interest in science. Some schools have classes with special curricula focusing on one or the other branch of science such as maths, physics or biology, etc. The best students are encouraged to take part in national or (the very best ones) in international competitions.A great tradition and perhaps one of the secrets behind Hungary’a excellence in mathematics and physics, is a journal written for high school students. This journal “Középiskolai Matematikai Lapok” (Mathematical and Physical Journal for Secondary Schools), widely known by its acronym KöMaL, publishes problems and solutions in maths, physics and informatics. KöMaL, founded in 1894!!, has helped to educate generations of mathematicians and scientists. KöMaL is now available also in English:  

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?
A: All of us are the intellectual products of our education at home, at school and at University. These have combined effects and I cannot identify any single piece of influence in this regard.

Q Which Nobel Prize or landmark piece of work, do you think has had the greatest impact on Life Sciences and which one on your own career?
A: Even the greatest discoveries have preludes and grow out of previous results, thus it is somewhat distorting to select just one. Yet, I think the Watson-Crick model of DNA stands out. The implications of the double helical structure have defined the framework of molecular biology research. 

Pál Venetianer
As to my own career, it was the discovery of restriction enzymes and DNA cloning that had the greatest impact. When, with a fresh diploma from Debrecen Medical School and finishing a 6 month compulsory military service, I joined the group of Pál Venetianer in March of 1975, my first tasks were the purification of restriction enzymes and setting up recombinant DNA methodology in the lab. At that time only a handful of Type II restriction enzymes were known, and DNA cloning just started to emerge after the landmark PNAS paper of Cohen, Chang, Boyer and Helling in 1973. Early during this work I had the good fortune to discover a new restriction enzyme (BspRI), it was my first paper: Kiss et al. (1977) Gene 1: 323-329. I have kept an interest in restriction endonucleases and DNA methyltransferases ever since.

Rich Roberts at Cold Spring Harbor
Q Who personally has made the greatest impact on your career in Science and how?

A: I can name two individuals, Pál Venetianer and Richard Roberts. I started my research career in Pál’s lab in Szeged and spent three years as a postdoc in Rich’s lab in Cold Spring Harbor. I am most grateful to both of them for the generous support, for the freedom to pursue my research interest and for teaching me, by example, devotion to science.

Q What do you consider your greatest achievement to date in Science?
A: The first application of the so-called methylase selection (originally suggested by Mann et al. in 1978, Gene, 3: 97-112) for the cloning of DNA methyltransferases (Szomolányi et al. Gene, 1980, 10: 219-225) and for the cloning of complete restriction-modification systems (Kiss et al. Nucleic AcidsRes., 1985, 13: 6403-6421). The vast majority of restriction-modification systems that have been cloned were cloned using this simple and powerful selection. The method was also used to isolate mutants of DNA methyltransferases that had altered specificity. At the 2nd New England Biolabs meeting in Berlin, in 1990, Tom Trautner called the method the "Hungarian trick”. I was of course very pleased to see that later some papers referred to the method by this designation.

Q How important do you consider maths and computing for Life Scientists?
A: It depends on the particular field of life science. As to my own field of study, I very often have the feeling that I should know much more maths and computing than I do.

My thanks to Antal and I hope we will welcome him to the UTC in the near future to talk about his work on restriction and modification enzymes.