genetics

This is what it has been. An initiation. I had crossed closed doors into a hidden world, a world that many will never see. More than that, I had crossed the border into understanding, and into seeing what lies beyond today’s knowledge. It was a Rite of Passage that changed me, that made me see the world differently. Right there, in the lab, I saw the genetic code of life transcribed, read and re-written. All this left me with a feeling of profound fascination, a sleek current of inspiration through my mind. I felt the call of the Odyssey. But above all, I saw people. People smiling, laughing, thinking, making it happen. It dispelled all the horror myths that orbit modern science. It also let me have a good laugh when I saw ‘Evolution’ in the cinema.

We are not people of the Matrix. We do not play games with the lives of men. We do not hold the key to the future.
We are the future.

A tight square of blue uniforms bunched up in the front of the hall. Thoughts orbiting the exam room, we hardly notice our Head of School saying something about Work Experience forms. When asked by my friends, I casually say mine is at the University. Biosciences. Nothing exciting. Everybody knew I was for some reason keen on biology. What I didn’t say was where – the Laboratory of Molecular Genetics.

Engulfed by the tide of exams, work experience seemed far away, once a sunny week in July (if sunnyness was an option). But as the day drew nearer so did the feeling that by now was becoming very familiar. The first time I felt it was when the plane took off for Australia, leaving behind light-dotted velvet night that closed around my past. I did not have a clue where I was going. The future did not exist in forevision – it was a blank, I simply did not know. Very much same here, five years on, as I stand before the office door – perception distils to the split-second present. I feel the unfamiliar smell, see the light slide on the pale corridor wall, read the letters that spell “Prof. C. P. Thompson” on the dark-blue door, hear the sound of my knock and my heartbeat. Somewhere on the edge of my consciousness is a picture of an ancient bearded professor – it promptly ran away to hide at the back of my mind as the old wooden stairs gave way to the very modern well-lit corridor, and now peeps cautiously round the edge.

I had tried to imagine what he would look like, all the guesses falling short of reality. A moment later my hand is clasped in a firm and energetic handshake, and very keen light grey eyes meet mine. A smiling, sun-tanned face, short-sleeved shirt. Later I would figure out he looked exactly like David Attenborough (come to his office for a break after chasing lizards up savannah), but at the moment I could well believe he came from the Himalayas.
There was a cool confidence there, and great seriousness too, but as I stood face to face with the Editor-in-Chief
of an international microbiology journal I half expected to see a eucalyptus tree outside instead of grim British skies.

The desk curves like a massive tusk as he puts his papers aside.
My preliminary lecture on genetics is about to begin.

An hour later I am sitting in the same chair with a thick book of safety precautions, wading through descriptions of semi-familiar or totally unknown equipment far beyond the boundaries of a school lab.
Agar plates. Micropipettors. Syringes - disposed of by incineration.
Do not re-sheath needle you might prick your fingers (why are we allowed to use them in the first place?).
Centrifuges – sealed plastic containers symmetrically balanced.
That does not ring a lot of bells until I see one programmed for a force 50,000 times earth’s gravitational field.
In my head a buzzing excitement from the ordered chaos of the lab. A formidable sight for the first time, each bench supporting a shelf gleaming with an armour of glassware.
First impression: WOW. (Second: oh dear…).

I had been introduced to a stack of manuals for the equipment and then to each member of staff whom Prof.
Thompson could grab on the spot. In the end I remembered names and titles with only a very hazy idea of whom
they belonged to – at coffee break it is down to intuition. Kasia, with golden ponytails and a charming Warsaw smile.
Jo, a gentle but serious English lady. Wei, a qualified surgeon from China doing his second degree, whose accent I struggle to follow but whose warm smile is reassuring. And my fellow spirit, Irena – British-born Ukrainian.

A moment later, when we emerge victorious from the battle with the drinks machine, looking gingerly at the synthetic-coloured fizzy liquid, we are joined by Tony, the computer whiz of the lab, and in another moment by ‘Sunshine’ Sian, an endlessly cheerful imp from the Welsh moors.

Tony looks up at Sian, eyes wide with child-like innocence. Carefully trying to sound serious, he replies:

“Um. It looks…” Tony’s eyes are sparkling in anticipation. “…Different.”

We are all laughing by now. Tony’s hair is bright blue. In addition he has just had a hair cut, so it bristles in all directions as if locked by electrostatic. That allows us to admire the fact that in a not-so-distant past it was bright green, as the roots still retain the last colour. Tony is revelling in the sensation he is creating. Two weeks later he will leave me a charming message:

Next is a visit to the teaching lab – a completely different world.
Sure enough, there’s a stacked prep room nearby, but the lab itself looks almost desert-like in its immaculate tidiness, the same marble-topped benches and sinks apparently spotless. On one of them today is a monolith of a machine, looking disconcertingly like something from Star Wars. By it is a more traditional-looking professor, with round glasses on a round face, untidyish mousy hair and not-so-spotless unbuttoned labcoat, who has possibly just climbed out of the spaceship’s research compartment to give us a demo of his mastercraft. We perch on a makeshift congregation of chairs round the bench, listening. The machine, as it turns out, is a Gel Imaging device that allows high-quality photography of electrophoresis gels, linked to a computer with state-of-the-art software for quantitative analysis. The question is the usual one: “To buy or not to buy”.

Looking around, it is interesting to see some people more confused with computer operations than I am. The software is brilliant, and, as such, extremely user-friendly. In fact it reminds me of some of the graphics packages we looked at in Australia. The lid is up, allowing us to see inside the machine, as a ray of light slides across the gel tank and we are told that exposure is varied by changing the speed of the ray. The printout of the photo on light, smooth plastic film is superb (in my journal it is the best, although the least informative, example of gel photography). It is passed around. Prof. Thompson watches with interest. My eyes flick round the rest of the group and alight on an absolutely classic ‘student from Kiev’. A strikingly handsome pale young face with a curly beard, mischievous and engaging eyes with a hint of green and a wild mass of darkish curls together with a bright blue shirt (the only touch of vivid colour in the lab save for dark-blue doors) make it half-impossible to believe he hasn’t just stepped out of a fine illustrated edition of “Fiddler on the Roof”. I wasn’t far off – Maciek, as he is later introduced to me, is Polish.

Demo over, and back in Prof. Thompson’s office, I am informed that a lab meeting is held on Mondays at one o’clock – an opportunity for everyone to increase awareness in the field as each week somebody takes it in turn to talk about an interesting paper they read recently. With a smile, Prof. Thompson tells me that this week the lot is his, producing a bookmarked journal from the depth of the paper piles. That would be in half an hour, and I promptly take the opportunity to disappear, saying I would prefer to have lunch now if that would be OK. Leaving the professor to read the bookmarked paper, I confidently head down the (wrong) stairs.

After the meeting, when I had finally decided that learning Japanese would not be that difficult after all, as by the end of the week I’d have a fair practice at listening to a language that might as well be alien and picking out grains of familiar words, there came a soft knock on the door. It is Jo, come to talk about her project. Prof. Thompson politely enquires if it’s all right if I listen as well.

The short sentence that followed I cannot attempt to reproduce, as it went completely over my head. I must have looked more bewildered than I thought I did – Jo, after one glance, opened her massive folder, leafed through a sheaf of diagrams and, settling on one, started a quiet explanation about how to make bacteria with one gene missing. Later on it would make a whole lot more sense.

My last task for the day was creating databases for genetic sequences of bacterial samples from Belarus and sequence comparison. Prof. Thompson was pleasantly surprised that I was quick on the computer (my fair competence with IT impressing him more than my knowledge of genetics, or rather the lack of thereof). While he was on the phone I patiently plodded through sequence alignments of the replication regions of the different strains. There were about 20, and each had to be compared to the others. Half way through, not impressed with quite frequent similarity of 40%, he asked me to print out the original word document. The sequences were typed in paragraphs, each a 300-base stretch without breaks, and at first glance shifted and shimmered like B. Riley’s abstract line paintings. I didn’t see how having the printout could help.

Within about 30 seconds of looking at them, Prof. Thompson quietly said “Check those two and those two”. My fingers obediently tap-danced on the keyboard. 98.5% and 75.4% identity respectively. My jaw quietly dropped lower and lower.

My Risk Assessment sheet, handed to me at the end of the day and listing the various activities that lay before me made quite alarming reading, with my mind playing back the professor’s words: “I expect you to not only learn the technical procedures but to understand the biology behind them”. On my way home I vowed I would study hard. Therefore I dutifully copied the structure of DNA bases into my journal and even attempted to do some reading in spite of the page blurring as I drifted off to sleep. Only when snuggled under a fuzzy quilt it dawned on me where I had been and what I had been doing. The words drifted in my mind, curling into little whirlpools of sleep:

“In the beginning was the word. The word proselytised the sea with its message, copying itself unceasingly and forever. The word discovered how to rearrange chemicals so as to capture little eddies in the stream of entropy and make them live.* …in the Beginning was the Word. …in the beginning…”

The next day introduced me to Richard. Somebody I soon found to be amazingly sensitive and intelligent. Somebody who would later associate in my mind with two images. One, an imprisoned prince. The other, a blue-eyed spaniel. He seemed very shy of attention, his speech defect becoming more apparent with agitation. The dull-coloured patterned shirt looked almost like moths’ camouflage, deflecting the limelight, yet at the same time making him subtly different. The only striking feature in his appearance were his eyes. Eyes incredibly blue and incredibly deep, eyes that so much stood behind. He was often alone, and almost equally often the butt of a good-natured lab joke. The main battlefield was between him and the loud, laughing Kasia, a contest the lab watched with interest and often participated in from behind the safety of the glass-armoured shelves. The chief coveted prize of the tournament – The Fan.

Sunnyness was more of an option than I had bargained for. It was not hot, which doesn’t happen in England, it was boiling. The lab was not equipped for extreme weather conditions triggered by the impending end of the world – in England the builders, watching the eternally overcast sky, had not thought about air conditioning. For the two weeks the temperature in the lab regularly soared to 29 and above, and we all silently cursed the microbiology labcoats – or the clothes underneath. The fan that Richard brought to his bench was fast proving the only salvation. A microbiology lab does have its advantages – pouring ethanol over your hands and holding them up to the fan is a wonderful way to cool off. The problem with that for Kasia was that she worked three benches down. Therefore it was not infrequent that if Richard arrived late, he arrived to an empty windowsill. The fan’s whereabouts didn’t take much guessing, and the shout “Kasia. My Fan!!!” heralded the start of another day’s battle.

“Richard, it is not your fan (not any more)!!! You should share it!!!” And, one day, “Let’s compare: two shoes, two socks, pants, undies, shirt, I have a bra you don’t so there I’m more hot than you I have the fan!!!”. Richard couldn’t argue with that – for the day, the fan stayed at Kasia’s.

One of my tasks for the day was monitoring cultures of Richard’s mutant bacteria. He had inoculated overnight cultures which could be harvested for analysis, which meant that their optical density had to be checked every hour or so. That entailed a brief trip down the corridor to the hot room (37°C) with cuvettes and a micropipettor for getting samples. After he showed me the first time, I convinced Richard that next time I could go by myself. Sliding the heavy door shut, I found myself in a humid environment not unlike the Australian rainforest, but the low buzz here was not the cicadas but the steady labouring of the vibrating platforms on which, clasped in spring-bound ‘claws’, yellow growth media populated by bacterial colonies swirled in conical flasks. After walking around them like a cat round the fish tank, tail swishing, I found a way to extract my flasks from the ‘claws’ (which consisted of determinedly grabbing hold and pulling hard). After transferring them to a stable ledge by the door I proceeded taking samples, when the door opened. That was when I discovered the ingenious use of surface gravity by the cuvettes’ inventor – the liquid does not immediately pour out of one when you jump and drop it upside down. It also prepared me for the next time the door opened and Richard came in with an apologetic grin – he just realised he forgot to instruct me on how to take the flasks out of the ‘claws’. I flashed him a proud smile – I was at least as intelligent as a cat – and stalked out of the room with feline grace, leaving him to close the door and switch off the light.

The major task that day, however, was SDS-PAGE, polyacrylamide gel electrophoresis of denatured proteins extracted from the culture samples. After assembling the gel tank and leaving the gel to set, Richard took me to his office to explain his project. He was investigating the mechanism by which the two replicas of the chromosome are partitioned and transferred to opposite poles of the cell at division. In 1992 researchers had identified two genes as a possible factor: one coding for ParA, a motor protein which could provide energy for chromosome partitioning, and the other coding for ParB, a DNA-binding protein which could attach ParA to the chromosome. At the moment, Richard was working on developing a way in which ParA and ParB could be visualised in bacteria under a microscope slide so their function could be observed. The day’s work was to separate proteins of different sizes through a gel matrix of fine fibres by movement due to an electric current (smaller proteins move faster and travel further, forming a series of invisible bands) and polish up a technique for colour staining only the ParB protein by immunoblotting. To do this, Richard used samples from genetically modified bacterial strains, in which the ParB gene, ParA gene or both were deleted from the genome (and therefore no corresponding protein would be produced), together with a control wild-type strain.

I watched the chain of blue bands formed by a marker dye crawl slowly down the gel plate, fascinated by the magnetic movement as by the slow crawl of ocean tides I had watched so often, trying to visualise the invisible trail of protein bands that streamed behind it like a flimsy veil, faint ripples in the sea of chemistry. After the gel had run, the proteins would be transferred onto nitro-cellulose paper that could be stored more easily, again under current. I listened to explanations as I watched Richard assemble the ‘sandwich’ of apparatus, then lift the gel slide out of the tank, two glass plates holding between them the elusive transparent medium.

“You lift up the top glass and wash it, while the gel stays on the bottom one.”

If you are lucky. Watching, I saw a jellyfish-like blob crumple and plop into the sink. That’s kind of weird.

Looking closer, I was quite sure, if my eyes didn’t trick me, that the bottom slide was just glass with a bit of liquid smeared on. I prodded it with a finger to investigate.

Unbelieving, Richard looked closer. Sure, the top section of the gel was there, showing a fine rip at the bottom. The rest was gone.

Was, at any rate. With a look of a magician who thrusts his hand in a jar not quite sure if he’ll find a bunny rabbit or a cobra, Richard reached into the sink. Fishing around, he extracted a jelly blob. Spread out in buffer solution, the rescued gel did not look much the worse for it, only slightly dog-eared at the edge. The results turned out fine.

Being an early bird and not finding Richard in the lab next morning, I pulled a random instructional manual from the shelf, thinking to leaf through to pass the time. Unexpectedly, within a few seconds I was absorbed in reading – I had stumbled on an autobiographical article of the scientist who invented PCR. Polymerase Chain Reaction is the technique used to amplify genetic sequences – its elegant simplicity of obtaining unlimited copies of a particular stretch of DNA by varying the temperature of the sample in repeated cycles had won a Nobel Prize. The theory was ingenious, transforming a process that previously took a mammoth amount of work. Oligonucleotide primers (single-strand stretches of DNA several base pairs long) are artificially synthesised to match the beginning sequences (5’ ends) of the gene on both the leading and the complementary strands. Forget tedious labwork – now primers can be designed and ordered over the Internet, with smart programmes telling you how very brilliant, or very otherwise, your design is, together with a pack of helpful tips to avoid truly spectacular disasters (such as the ends of the primer, being complementary sequences e.g. ACA and TGT, sticking nicely together). The primers are added to original template DNA together with dNTPs (deoxynucleoside triphosphates) – ‘building blocks’ which contain a particular base and can be stitched together by a linking enzyme to form a DNA strand. From then on it is simple. At around 94°C, the two strands of the DNA double helix separate. Dropping the temperature down to 55-60°C makes the primers stick to the complementary sequences on the single strands of template DNA. Heating the sample to 72°C optimises the temperature for enzymes that quickly extend the primers with a sequence complementary to the template strand using free dNTPs.

At 94°C the newly synthesised strands separate from the template, and the process can begin all over again as the temperature cycle is repeated. Furthermore, the primers can now stick to the newly formed strands as well, so the amount of product after each cycle increases exponentially as the power of two. Starting with a single template double helix, after 20 cycles you would get over a million of replicas (2²⁰). If more primers stick to all the single strands formed, by the next round you double the number. The stroke of genius for the process is the use of two primers, which means that the vast majority of the product will be exactly the length of the sequence you want, as the primers would alternatively define the ends of new strands. Of course long sequences are a problem, as the probability of random mistakes grows, but anything around five thousand base pairs works fine.

And none of this would be possible if volcano-living bacteria had not given humanity a helpful hand – provided enzymes that can withstand temperatures of 94°C and above (enzymes in the human body denature at about 42). The British love to take the credit for the first isolation of such enzymes from bacteria down at Yellowstone National Park. Anyway, it’s incredible how many of our scientific advances are due to the minute life forms we generally do not think of otherwise than harmful. A friendly thought considering Martians are probably bacteria too.

As Richard had not re-appeared, I wandered down the lab in search of adventures. Kasia, softly humming to herself, was doing ‘minipreps’ – isolating plasmid DNA from a series of mutant strains. This sounded totally fascinating – actual manipulation of DNA!!! Hands-on Genetics. The double helix is not just a picture, you can hold a sample of one in your hand!!! All this whizzed through my head as I hopefully asked if I could help.

“You want to do minipreps?!” Incredulous voice, her face lighting up with a smile. “OK. Label these tubes 1 to 12, here’s your syringe, it’s all yours!!!”

I would soon understand her excitement. A vivid imagination is helpful – you can appreciate the grandiose implications of what you are doing while your hand mechanically dispenses almost invisible quantities of alternating colourless solutions into 1ml microfuge tubes secured in a foam rack, the bottoms of which you can’t even see. Well, that’s not true actually. A vivid imagination is unhelpful, as I soon found out – with a syringe it’s fair enough, but with a micropipettor, changing tips and reaching into reagent bottle every time, it’s awfully easy to lose track of which tube of the row you have already filled. Not being able to see the bottoms is not a major nuisance, as I soon found out – the solutions being transparent and the quantity often so tiny (several thousandths of a millilitre) that whether you added it or not, it looks pretty much the same. That’s why, Kasia explained, giggling, we call the Chemistry department “bucket chemists”! In a Microbiology lab, one millilitre is a lot.

There is, also, no better place to appreciate molecular scale – Irena sets her PCR to 35 cycles, starting with lots of templates – all in 1ml tubes. I remember the sigh of Prof. Thompson as he looked down at the profusion of bacterial colonies, blending together like vast metropolises, on the agar plate I had streaked. I had picked a tiny scoop of bacterial culture with a fine metal loop only to smear a starter streak, then flamed the loop every time as I streaked the rest of the plate, briefly touching previous smears. “This is typical”, he said gazing down thoughtfully – “Perhaps students don’t realise from the start just how many bacteria there are.”

Following minipreps, I set up two restriction digests under Kasia’s instruction – reactions in which specific restriction enzymes are added to the purified DNA, which cut the DNA strands at specific sequences, “cutting out” the gene sequence you are interested in. After jotting down working concentrations in my journal, Kasia set off towards the lab fridge. Enzymes for lab use are usually kept at -20°C, although later I was taken down into the basement, where huge steel-clad fridges struggled to maintain a temperature of -84, a piece of the Arctic in a room which was noticeably hotter than the rest. While she picked out brightly capped microfuge tubes from the stacks, brushing off hoarfrost coating that hung down the freezer shelves like a shaggy mammoth hide, and transferred them to a tiny ice bucket which immediately grew a pearly cover of condensation, I stared at the elaborately coloured chart blue-tacked to the fridge door. Following my gaze, Kasia paused to explain. The chart listed enzyme activity in different buffers – as the different enzymes are isolated from different bacterial strains, they work at their optimum in different buffer conditions. Therefore if you use more than one enzyme in a single reaction, you have to find a ‘compromise’ buffer in which activity of both would be 50% or above. If the enzymes are absolutely incompatible – tough luck, you set up two separate reactions with a wash step in between.

After restriction, the DNA fragments are separated on an agarose gel, which works on the same principle as polyacrylamide gels, only giving a less precise resolution. I soon found out that loading samples into gel wells, visible only at a certain angle of vision, is nothing less than an art – half a millimetre out and you see a lacy cloud of dye-marked sample blossom out of your pipettor tip and diffuse nicely and evenly across the whole gel plate.

While the gel was running, I caught up with Richard – I was actually supposed to work with him.
He was proceeding to visualise his nitro-cellulose paper bound ParB, which involved successive application of antigens, antigen-specific antibodies, then antibodies to recognise the first lot, bound with a specific chemical, and more chemicals to produce a coloured compound, interspersed with lots of washes and periodic incubations of 1-2 hours. Once I grasped the theory the prospect of practice did not look very exciting, so I firmly headed off to the UV camera room to see Kasia’s previous gel.

UV-light visualisation of DNA in gel stained with ethidium bromide was terrific – the bands showed up a glowing sunrise colour, close to a warm apricot, on the background of night-sky blue. The black-and-white photograph was rather disappointing. Some results were good, two clear bands, for the cut out gene and the rest of the vector it had been inserted into. Murmuring “bad luck” after looking at the rest, she dug into her journal, considering which steps should be re-done. It was then I heard Richard call my name. Slightly guilty, I started towards his desk, only to find the whole lab gathered around a sink. Oh no.

Coming closer, I saw a beaker of vigorously bubbling leaf-green liquid. The honeycomb column of bubbles which rose from it soon bent over and flopped into the sink like a slinky. Laughing, Richard flicked at it with his hand. Some of the bubbles popped, releasing curly wisps of white mist; what remained had a very spectacular look of a column of bubbles with a piece bitten out of it. The sight was completely bizarre – it looked for all the world like they had a flame-breathing dragon trapped under water. The lab, a cabal of white-clad alchemists bending over their bubbly green creation, was immensely enjoying my dumbfounded look of “What the hell is that?!”

In the spirit of a true investigator, I dared to touch it. It felt very cool. Double, double, toil and trouble – I did not have a clue of what it was.

At the end of the day and back in the office of Prof. Thompson, I reported, smiling, that it was twenty past six – i.e. well past my scheduled ‘leaving’ time. It was a lab joke that my days were getting progressively longer. I soon made a surprised discovery that he knew as much about my day’s work as I did, and was quite aware that I had watched the cherry-brown staining of Richard’s protein as well as doing minipreps and gel for Kasia, gel-cleaning and setting up insertion reaction of a GM gene into a vector for Irena and pouring agar plates for them both. He had also privately given up trying to ‘plan’ my activities. From here onwards, he would just ask “So what are you doing tomorrow?” and then casually enquire whether I understood all of what I just said.

First on the schedule for Thursday was XylE Assay with Kasia. Not quite convinced by my brave reply that Kasia had explained to me briefly what it was all about, Prof. Thompson proceeded to give me a more fundamental lecture. The technique involved regulation of gene expression – as the vast majority of the cells in an organism contain a full copy of the genome but do not need to express most of the encoded characteristics, there is an operating system of gene regulation. Each gene contains a ‘promoter region’ at the beginning of its sequence – a site at which DNA transcriptase, the enzyme that decodes DNA into RNA, can attach to the DNA strand. Negative gene regulation involves the production of protein that stops transcription of a particular gene into RNA by either blocking the promoter region or by binding to and inactivating transcriptase. This was one of the functions of KorB, the protein that Kasia was working on – it stopped the transcription of XylE gene. Therefore, the effectiveness of mutant types of KorB could be assessed by assessing the activity of XylE. The XylE gene normally codes for an enzyme that oxidises catechol, turning a transparent solution yellow. Therefore, the activity of mutant KorB could be determined by measuring how yellow the solution turned on a spectrophotometer. This is followed by checking the total amount of protein in the sample – seeing how purple the solution turned with the Buiret reagent. Colour chemistry being my favourite kind of magic, I happily spent the morning scanning yellow samples on the ‘spec’ and turning them purple.

Just as I finished Irena came up with a sheaf of photocopied notes. This left me with a very familiar school task – cutting, pasting and reading through a welter of technical information on ligation – insertion of a gene into a bacteriaphage vector that can then be used for transformation (infecting a strain of bacteria with the vector and so causing integration of the gene into the bacterial genome). Ligation may look complex, but transformation is certainly fun, involving pouring both samples on an agar plate, adding some glass beads and vigorously shaking by any preferred method (including jumping up and down if desired). Inspired, I proceeded to carry out the operation and quickly confirmed the First Murphy Law of Science: Nothing is as Easy as it Looks. My sterilised glass beads were happily rolling all over the bench and floor.

Having chased the beads under a bench I decided to check on what the rest of the lab were doing and bumped into Lewis. He was setting up a PhastGel – automated version of the polyacrylamide gel for proteins, in which gel slides and buffer strips come ready and packaged. An easy solution, but with one setback – for twelve samples the gel slide is about 4cm², so loading the gel is a jeweller’s work! There is a plastic slide with tiny round wells, 2mm in diameter, which are covered with sterile plastic tape, and onto each you drop 2 microlitres of sample. The drops – minute, perfectly round sapphires as fragile as dew-drops on a cobweb, held apart only by surface tension – are picked up by a comb, the teeth of which have tiny slits that draw in the samples by capillary action, and are lowered into the gel. After a while, you can see a silk strand of bands migrate slowly to the opposite pole. The resulting gels are so small they are stored in petri dishes – the resolution is nowhere near detailed, but it’s a quick way to check you’re dealing with the right protein!

With the next round of minipreps for Irena I started to get my head around the whole process by which bacterial strains with specific mutations are produced, or I should rather say handcrafted. The most intriguing step, for me, has to be the DNA sequencing, the actual process by which we can read the Word that started life. The process we used was the Dideoxy Sequencing. It is built on a principle of new strands forming from a template DNA by linking dNTPs, as in the PCR reaction, only this time with addition of ddNTPs. When a dNTPs (dideoxynucleoside triphosphates, hence the name dideoxy) is randomly incorporated into the new strand instead of a dNTP, the strand terminates as ddNTP can form only one bond. Therefore, the products of an “A” reaction with ddATP addition will include a randomly accumulating range of strands of varying length, each of which begins with a known primer sequence and ends with an “A”. Setting up four reactions, with ddATP, ddTTP, ddCTP and ddGTP, and separating the products according to size on a high-resolution polyacrylamide gel (stained with radioactive P³²) will result in a consecutive series of bands in the four lanes, from which the sequence can be read.

The automatic sequencing device is even more ingenious.
It uses liquid gels of ultra-high resolution, which are pumped into 96 tiny capillaries and set under pressure, enabling 24 sets of sequencing reactions simultaneously.
Fluorescence-marked DNA is detected by a laser beam as it is carried out of capillaries on currents of buffer solution, as capillary walls would reflect some light and lower precision. It is mindboggling to think that nothing physically separates the DNA-carrying currents except the speed of flow, but that precision is almost infallible. The amount of fluorescence detected is plotted in a graph and when the graphs of four reactions are overlaid they form a series of peaks of alternating colour, from which the sequence can be read. The printout is exactly what the Human Genome fragments looked like in the Nature publication.

Next day I saw two very different sides to genetics. The Underworld (may I be forgiven for the nickname, the staff were all exceptionally nice!) was in fact a floor up. This was the place where all the basic solutions were made up, all the growth media were prepared, sterilised and bottled, all the imported disposables sterilised before use and all the biohazard waste was disinfected before disposal. With some equipment Lucifer would be proud of, I thought as I stood before the ‘dirty’ autoclaving machines – gigantic pressure cookers, thick screwed-tight doors containing an inferno of 121°C at twice the atmospheric pressure. And smelling as only waste autoclaving machines can smell.

In another room, great metal hulks of the ‘clean’ autoclaving machines overlooked ordered shelves stacked with boxes of plastic micropipettor tips sealed with autoclaving tape, a kind of masking tape with slanted white stripes that turned black when autoclaved. Nearby, a steamer for heating and melting growth media before it is poured into agar plates, with a pair of thick felt gloves. Prof. Thompson smiled as he instructed me how to lift the lid, allowing a cloud of steam rush out in a solid wall:

Coming down into the lab, I was presented with an imposing volume on clinical microbiology detailing composition and use of various growth media, techniques for sampling and inoculating bacterial cultures and ways of identifying known pathogens, which I immersed myself in until the medium melted.

The second tour was to the “Stars” of Genetics – the Functional Genomics Lab. Located in the west wing in a zone of restricted access (no undergraduate students!) and eternally closed to the ‘uninitiated’ (the only place requiring swipe card access at any hour of day), the laboratory houses 3.7 million pounds’ worth of exotic equipment, and, from time to time, distinguished visitors in black suits. Well-lit and, oh miracle, air-conditioned, with colourful posters advertising gold-plated slides and exquisite microequipment, and still more colourful price lists on the walls, with computer technology that could rival the poshest Microsoft research labs and apparatus the function of which strained the bounds of credibility, it was a place where all I could think was:

The atmosphere of the place is beyond description. I can only describe some of the things it does.

By the far back wall, a row of mighty PCR machines, each the size of a fridge as compared to our lab one that comfortably fits on a bench, and completely automated devices for rapid DNA purification. Joining the rank is the sequencing apparatus, with a transparent-walled fridge for storing sample trays. Closer to the entrance is an imposing scanner that detects radioactive labelling – hence the massive radiation hazard sign on the door. And, in a compartment of its own, the MicroGridII – a mind-blowing device for screening gene expression. Special trays are loaded with separate amplified sequences of each gene in a genome (easily possible for E.coli, the bacteria we worked with, as it has only 4000 genes, or part of a genome for a more complex organism). Samples of the genes are then picked up by tiny needles, and transferred to a microscope slide, forming a microgrid of dots in a space of 15mm². The grid is fixed and placed into a solution of fluorescence-tagged RNA isolated from the cell, which binds to the corresponding genetic sequence. The slides are then screened under UV light, with genes that are expressed under given conditions and therefore have matching RNA lighting up as a series of dots. This gives a literal map of a genome that can be used for monitoring, for instance, which genes are expressed under particular stress. The eyes of Prof. Thompson glowed as he explained the potential of the device. His enthusiasm was catching.

Across the aisle, Maciek was screening his radiation-tagged protein gels which assessed the DNA-binding activity of mutant KorB in the presence of another protein, IncC. Next to him, Bloxy, the tall computer technician of the lab, was adjusting something on one of the screens. Quickly grasping the opportunity to stay in the lab a bit longer, and in the company of two charming gentlemen, I innocently asked the professor if what he intended to do with me in the morning could wait and if I could watch this. With a knowing smile he replied that yes, I could come and get him in his office when I was through.

By the time I ‘was through’ it was lunchtime, followed by another lab meeting. This time it was more serious, with each member of staff reporting in turn on the week’s progress, back in Prof. Thompson’s office – rather a squash for the twelve of us. It was a curiously intimate feeling as we sat in a tight circle facing each other across a few feet of empty floor, a conclave of explorers discussing our secret business away from the eyes of the rest of the world. I can feel the barely perceptible difference in temperature on the inside and on the outside of the circle, as I listen to the quiet hum of conversation. The whole scene has a mystic air. I am sitting in the middle of something that is not for the sight of strangers.

And the air intensifies as I see Prof. Thompson take a small key, open a drawer and take out… a deck of cards. At first glance I think they are Tarot. He shuffles them with an expert magician’s hand, looking, with his swarthy face, like an eastern shaman. Everyone is silent, and you can just hear me thinking “What is happening?!” He reverses the deck and spreads them out with an elegant sweep of the hand.

In silence, the lab picks a card, exchanging meaningful glances. I hear a voice:

Make Jane do what?! Someone whispers “This is an initiation into the lab”. Uncomfortably feeling all eyes upon me, I pick a random card. It is the ten of hearts.

Prof. Thompson picks a card too, walks over to his desk, takes out a pen and a chart.

A groan from Wei, “Oh no!”. We all simultaneously burst out laughing. He’s got the ace of spades.

Prof. Thompson surveys the open cards. “Right. Top of the list. Wei. Washing floors. Lewis, disinfectant. Irena.” Smile: “ Sinks. Kasia… oh no, we’ve got Jane. Right. Cleaning centrifuges. …and” – looking at his card, five of diamonds: “ I’m next week’s supervisor”. Sian voices the common opinion, “What a surprise.”

Next week I, a fully accepted member of the lab, bearing the honorary emblems of gloves and ethanol bottle, survey the row of centrifuges…

This week and the next flew by, a flurry of lab tasks, reactions, notes, tip boxes, and endless rows of ACCCTGACT…waiting at the bus stops while recapping the day’s lectures, excited stories to my parents and e-mail notes to my friends about Genetics being WOW!, skim-reading background research papers, charting amino acids in my journal, falling asleep on the next page with the light on, endless excitement and challenge and happy exhaustion. To some it may sound like I’m such a major loser but that is so not true. I had discovered a whole new world. I had also discovered nights were too short.

What stands out brightest from the next week is my professor’s look of mild shock when the reply to his “Is there anything else that you had expected to do?” was “Yes, I would like to try my hand at reviewing a scientific paper”. His piercing stare as he leans back in his chair, crosses his hands and enquires what do I mean by a review. All that is in my head is “Sir, I kind of hoped you could tell me that.” Soon, I gaze at the selection of journals as he offers me to take my pick, and, daring a diplomatic move, lay my hand on the journal he is the editor-in-chief of. Scanning through titles and abstracts for something that hopefully might sound vaguely familiar, I choose a likely paper to try, and settle into reading. Quite amazingly, it makes rather a lot of sense, and I can even follow their techniques. Politely asking if I could borrow the journal, I make my way to the lab. It is a relief to be out of the office.

Next day it is the visit of my teacher. It is interesting to see her bewildered look as she is shown into the lab by one of the university staff – the lab is quite a labyrinth, and looking like there might be more than one Minotaur. I hasten to meet her, Lewis takes my labcoat and we meet Prof. Thompson in his office. Assuring him that the interview is not inquisition and I am allowed to be present, she asks him what I have been doing. Professor quietly replies I’d better tell her myself. After listening to my attempt at brief listing of techniques, and waiting to make certain nothing more is going to follow, he informs my rather startled teacher that “She is writing a review of a paper for me.” He adds, with a half-apologetic smile, that it was my own initiative. He is not a slave driver. He had also never been asked that by a work experience student before.

My teacher assures him that I do seem to be quite fond of genetics, and that it had confirmed my career choice. I did say that, and I meant it.

His wide-open eyes lighting up with astonishment and something more foxy. All I can think is “Oops.

Next morning I duly turn up with a printout of my ‘review’, fully expecting criticism. After checking if I had looked up the meaning of a few long words, and nodding, satisfied from my reply that I did, he plunges into proper reading. His face is extremely serious. He only laughs as he corrects the spelling of “tuberculosis”, as I murmur inaudibly it must’ve been a typing slip. At the end, all forthcoming comments are: “That’s fine, Jane. I think it is very good.”, followed by “Can I keep the copy?” and “Do you know what organism M. Leprae is isolated from?” I have not got the faintest idea. “Armadillos.” Oh. Um. How fascinating. I secretly breathe a sigh of relief, and wonder if he notices my heart pounding. But that is not all. After thinking for a moment, he reaches into a file for a printed sheet. It is some second year undergraduate problems. Since I am so interested in the ‘other’ aspect of science, would I care to try some? He can explain to me, of course, how to do it, but I might feel like trying to figure them out by myself. Saying I would and taking my chance to escape, I pick up the sheet and head towards the lab.

Irena was not there, so I happily settled at her desk. The problems did look a bit daunting, but I soon found out that once I grasped the principles of experiment (which was easy, as it was agarose gel electrophoresis of enzyme-digested DNA, which I had done already) the rest was just logical thinking – or intelligent guesses. Prof. Thompson was impressed.

I hastened to be out of the office. Anyway, I had another intriguing task – fluorescence microscopy. Slide-making was Wei’s domain, and on the sink by his desk stood a spidery piece of apparatus which the rest of the lab quietly avoided, just as they quietly held in awe his steady hand of a practised surgeon. He had shown me some of his old slides before, but today he promised me I would make some slides of my very own. And so very soon I was washing the slides with alternating solutions, perched on a chair in front of the bewildering contraption which was in fact a vacuum machine, one hand carefully controlling a micropipettor and another guiding a spidery tube to suck the liquid off the slide. It was a tricky task, demanding minute precision with both hands simultaneously and guided mainly by the light reflecting off the surface of the drop, made even more so when certain solutions had to be applied for exactly 20 seconds. Hand on timer, take new tip, take up solution (toxic), carefully and quickly cover four wells starting timer on the first one, dispose of tip, grab the vacuum tube and stop timer before it rings your head off. With perfect coordination that does take exactly 20 seconds, with coordination less than perfect it is supremely annoying.

The next day Wei was all smiles. “The slides you made me, they were very good! Wonderful, wonderful.”

I felt I grew up three inches in the estimation of the lab. To manage Wei’s slides was an accomplishment.

Looking down the microscope at fluorescent bacteria is like looking up a starry sky, except some of the stars are oblong. Patiently, Wei taught me how to switch filters and photograph alternatively under UV and normal exposure, guiding my hand down resolution controls in the darkness illuminated only by the flickering phantom glare of the computer screen. Watching the thin iridescent beam and listening to the faint heartbeat clicking of the camera as it adjusted focus, I wondered how galaxies and flaring supernovas would look down a telescope, the domed shutter of the observatory sliding down ever so slowly to reveal a white-studded blackness. This is perhaps the closest I could come to it.

On the last Friday heat wave mounted under glowering clouds. Electricity was in the air as I entered the now-familiar office. Prof. Thompson, leaning back, passed a hand across his brow. This is what it feels like, he said, the lull before the monsoon breaks. And then everyone, instead of hurrying inside, runs screaming into the rain. The Himalayas. Trapped in the old red-brick University I was thinking the same thing, of tropical rains crashing down onto a white shore. I held his gaze. At this moment, we were closer in spirit than ever before, and it was the time of parting.

It was the lab joke, started by Lewis when he saw me battling with a second sheet entitled BSM208 Bacterial Genetics – Test, November 1999, that after this I could graduate right now. It did feel like a graduation, that last day of smiles and hugs and best wishes. Looking now at the scrawled notes in my journal I see them all – Irena, my fellow conspirator and kindred soul, Kasia with her glowing smile as she asks if she can write me something in Polish, Maciek tugging his beard for inspiration, Sunshine Sian with her farewell message “Well done for surviving!!”, Tony and his blue hair, Richard and his blue eyes, gentle Jo, Wei and his strong, steady hand, and above all Prof. Thompson – an utterly extraordinary person I was honoured to know, whose signature is last in my journal:

The rain did come with the promised fury. Looking at the liquid curtain over our coffee, Irena defined for me what the two weeks were. A crash course in Genomics.

Two weeks. 336 hours. A change.
__________________________________
* M. Ridley, Genome: The Autobiography of a Species in 23 Chapters. Fourth Estate, London, 2000.

Yevheniia Mikheenko

July 2001

e-mail: mikheenko@btinternet.com