GFP Biolistics Bombardment, Onion Epidermal Peel

Although laboratory work comprises about 80% of my waking life, only a small proportion seems to be making it onto this blog. The main reason for that is because lab work is not very interesting to most people, so I try to keep the focus more on life in Korea.
But I found out through my over-performing Sitemeter that some readers are starting to find this blog through science related searches. Often times I'll try looking for a protocol on Google, and the only results that come through are too technical to be useful.


To that end, I've decided to occasionally put some of our lab work up here, in the hopes that some of it will be useful to a perplexed undergrad student somewhere.
The protocol that we're going through today is GFP biolistics bombardment (only the machine part). A protocol is the science name for the 'recipe' of an experiment, and GFP is a fluorescent protein (think of deep-sea jellyfish or glowsticks). Biolistics involves coating gold or tungsten bullets with modified DNA and literally shooting it into the heart of a living cell. It was pretty revolutionary a few years ago, but has since been eclipsed by Agrobacterium-mediated transformation. In the photo above, Chen-Jing is peeling onions, in preparation for the experiment.


But first, let's have a look at this list they've posted up at the biolistics lab at Sejong University. I'd say it pretty much sums up the working conditions of Korean graduate students.

Except that we normally work longer than that.


This is what bacteria looks like when you grow it in a test tube. Bacteria are obviously too small to see, but the liquid they're in will turn cloudy when there's enough of them. In a typical inoculation, a few hundred cells will multiply into billions in just 16 hours. So imagine a classroom of kids multiplying into the population of the world overnight.


This is the biolistics machine. It's basically a crude particle accelerator that uses helium gas to propel the charge downwards. Make sure you've opened the main tank valve and connected the vacuum pump securely. The device is remarkably simple to use, but don't operate it by yourself the first time.


The first thing you need to do is unscrew the large metal nozzle in the chamber. Use tweezers to transfer a sterilised rupture disk into it and make sure it sits flat. The purpose of the rupture disk is to hold back the air pressure to a specific point, like 1000 psi, after which it will burst and fire the charge. You can change the specific pressure by changing the value of the disk.

But why do you need to change the pressure? It's because some plants have thicker skins than others, and you have to make sure you get the pellets inside the cell.


Then you take out the tray that normally sits directly under the rupture disk. Take it apart and put a gauze mat in it. This protects your sample from getting obliterated by the macrocarriers.


Next, put your macrocarrier holder facing downwards onto the circular top of the tray. The macrocarrier holder is the brown plastic circle that has your dried DNA on it. Prior to this, you need to have coated the metal pellets with your DNA and dried it with ethanol. The black spot in the middle is the tungsten residue.


Then you screw the metal clasp on top. It should all fit together very simply, like a Meccano set.


Put the tray into the chamber and get your sample ready. The best thing to use for GFP is onion cells, because they're big and clear so you can see them easily under the microscope. Unfortunately though, they don't have chloroplasts (green things responsible for photosynthesis), which means you can't really extrapolate the results to situations in leaves. They also have rather large vacuoles (empty spaces) right in the middle of each cell, so you need to be careful when you interpret your photos. But anyway, they're a good starting point.

You need to peel the slivers of onion skin that come from the middle of the onion when you cut it. An onion is just layers of bulbs, like Russian dolls, all layered on top of each other. Take the bulb in the middle that is around half the circumference of the entire onion and you'll find it has a thin skin sheath around it (like all of them). Cut a little square out of that and put it on 0.5% Murashige and Skoog media.


Okay, so now you put the sample underneath the firing apparatus. Remember to take the lid off the plate.


Close the door tightly. Hold the middle vacuum switch down until you can see the pressure gauge reach 27. When this happens, quickly switch it over to 'Vent', and the value should stay constant. Then hold down the 'Fire' button until the secondary pressure gauge (the one on top of the vacuum chamber) reaches around 1000 psi. You keep holding it until you here a popping sound, which is the sound of the rupture disk breaching.


Release the button, remove the plate from the chamber and wrap it in aluminium foil. Store overnight for around 12 hours, in the dark at 28 degrees C. The next day, prepare microscope slides and dye stains of your samples.


And this is what you'll see in the microscope the next day if you've done everything right. Dazzling, bright and beautiful. Make sure you have the right filter set on, and you shouldn't have to search very far. Real GFP fluorescence jumps out at you in a very conspicuous way. If you find yourself squinting at a faint blur and wondering if it's a real signal or not, you might need to adjust your incubation time or plasmid concentration.


My first picture was nice and bright, but it was too intense to be useful. After a bit of fine tuning I ended up getting this photo above, which shows some nicer details of the onion cells. The green blobs are localisation positions of my fusion protein, and the blue colour is DAPI staining, which highlights the nucleus.
So basically, what you're seeing is the DNA of a glowing jellyfish protein, fused to a bacterial protein, that was then shot into an onion cell which managed to translate it. It's the convergence of three kingdoms of nature, brought together through biotechnology. Cool, hey?

If you're having trouble with your protocol, send me an email and some of your photos and I'll try and help you out.

That's all for me this time. Happy bombarding everybody!

An Umbrella in the Laboratory

I've just entered my third month of the Ph.D here and getting into the swing of things. It takes around six months to a year in most labs until you can work completely independently. This is because there's a lot of know-how involved and the fine tuning of your methods really means the difference between an experiment working or failing. Some experiments take a few hours to do, but the more complex ones like a Yeast 2-Hybrid can take a couple of weeks in their entirety.

One feeling that you get used to pretty quickly is the one that comes after realising that you'll have to repeat a week's worth of work because you didn't get the results you were looking for. These troughs of feeling are compensated by the highs that you get when your experiments work nicely. When your experiments work, you feel like an invincible genius.
In the photo above, Chen Jing is cutting out a band of stained DNA with a razor blade. The gel that the DNA is sitting in is fluorescing pink. If you stain DNA with the right stuff, it becomes fluorescent under UV light. That's also why she's wearing the face shield, to protect her from skin damage.

On the left is Se-kyung and on the right is Hoon. Hoon is my senior in the lab and I have to learn a lot of methods from him. However, he is very 'traditional' in a Korean sense, which I would best describe as displaying a rigid adherence to a hierarchical social structure to every last detail. Korea in general does have a more formal system of etiquette and respect based on age, but Hoon is a little more intense than your average Korean. His demeanor as well as his large fingers (which can make the intricacies of scientific work difficult at times) leads me to believe he may have been historically misplaced and actually belongs to the age of gladiators.

We're learning to get along with each other as time goes by.

In the last photo you may have noticed Se-kyung with an umbrella. When I first saw this here I did find it amusing, but since taking this photo I've seen it enough times and also had to use it myself. When you need to take a photo of something scientific, you need to do it carefully. Everything in the frame has to be lined up and zoomed in to the same measurements, on a tripod and with an umbrella to shield against reflections.

Last week I attended a 2 day safety course with a lot of other new students. It was all in Korean, which meant I only understood about 5%, but it was interesting to observe the lecture room behaviour here. Korean students will often put their heads on the table during lectures and fall asleep (they even have a special verb for it: opdeurida). The lecturing professors, who I have to admit do appear boring even by lecturing professor standards, just continue on with the lecture like everyone's listening.

There was a test at the end, which was fairly straightforward. The content was a little silly at times, with such worthy safety advice as "If there is an unlabelled chemical on the bench, do not taste it" and "Don't store your lunch in the hazardous chemicals cabinet."
Safety education is vitally important to lab workers, but perhaps they also need to be testing us for common sense.

We do a fair bit of recycling here, which is good for the finances as well as the environment. I was mildly surprised to learn that they even recycle the toothpicks that we use to spot bacteria onto the petri dishes.
But I recently found out that this isn't done to save money. Do you know the two kinds of toothpicks that there are? The nice ones have pointed ends, while the cheaper ones are roughly cut and have a semi pointed end and a thicker one. The cheaper toothpicks are actually more useful to us, because the thicker end is a perfect size for scooping up a colony of bacteria, but the nice toothpicks are too pointy. In Korea they only sell the nice toothpicks, so the lab ordered some cheap toothpicks from overseas. Because they're not easy to come by here, we recycle them.

If you're thinking of a new business idea, try selling cheap toothpicks to Korean labs.

Earlier I mentioned one of the longer experiments called a Yeast 2-Hybrid. The theory behind it is fascinating and elegant, but getting it to work can be a nightmare. Basically it's a way to test if two different organic molecules interact with each other. Molecular interactions are behind almost every little thing that goes on in nature; from growing hair to producing acid in your stomach. By figuring out which molecules are interacting with each other, we can draw up a diagram and get an idea of what's happening in the bigger picture.
What a Yeast 2-Hybrid does is manipulate yeast cells so that they will live or die, depending on which molecules are interacting inside them. A good example of this is 'poison resistance', which is when the yeast can only survive poison if they have the correct interaction going on. In the photo above you can see that most of the yeast spots are nice and white, meaning that they survived. But there are two spots that are opaque, which means that they were unable to survive the poison. This tells us something about what is going on with the molecules that we are studying.

And in science, one experiment is never enough proof. What you need to do is repeat the experiment until you can convince your professor and the community that your results are meaningful. A good way to do this is to have experiments with different methods. If your experiments have different methods and use different stuff, but the results indicate the same conclusions, then you have a stronger basis for making a claim. This is why real scientists know that things like global warming and evolution are beyond reasonable doubt. These two 'theories' are supported through an immense range of scientific disciplines, from molecular mechanics to ecology and atmospherics. When such broad, independent and scientifically reliable sources all conclude the same thing, you can be confident that it's more trustworthy than someone who wrote a book saying it's all a big conspiracy.
Anyway, in the photo above is the same experiment but done with different chemicals. In the dish is essentially the same colonies of yeast, but they've been given chemicals that will turn blue if the result is positive. We can see that the same pattern emerges as in the previous photo, which gives us a more trustworthy result. Isn't life grand?

It was Se-kyung's birthday this week, so we celebrated with a cake and some Chinese food. Se-kyung is finishing up her master's degree this year. It's going to be Heather's birthday next week, so I'm probably heading back to Busan for the weekend.

Here's a fried fish cutlet lunch that I had a while ago. The serving sizes are actually quite good and just enough to fill you up without going overboard. If you want more though, you can go back to the counter and get refills for free. The price of this meal was only W2,500 (about $2.50 Australian!).

It was also graduation day recently. A whole lot of vendors came to the campus selling flowers, food and photography services. I look forward to the day when I graduate, but I guess I have a lot to learn before I can consider myself proficient in the field.

The professor took us out for a nice dinner last week too. We went to a traditional Korean barbecue place to welcome our newest lab member, Keonwoo. One day I'll hopefully get a photo of the professor to post here. He's one of the smartest people I've ever met and quite friendly, but I still haven't struck up the courage to snap a photo of him. Maybe one day when he's not looking, you'll get to see a photo of the back of his head.

I'm still on the waiting list for the dormitories and my old place expired. So Hong-sup organised some new accommodation for me at the New Zen koshiwon. These types of places are popular with students because they're cheap and usually conveniently located. Mine is 30 seconds away from the shuttle bus stop.

Koshiwons have shared facilities like kitchens and washing machines which give them a more communal atmosphere than regular lodgings, known as officetels.

And here's the reason why they're so cheap. My room has enough standing space for one, but not enough floor space to do push-ups on. But it helps to keep the place tidy at least. I have free internet and a little TV too. Some people live permanently in these sorts of places, and I'm sure I could live here for a year or so. It's not so good for having friends over though.

My room is one of the 'deluxe' rooms, which means I don't have to share my bathroom. The showerhead actually sprays over the whole bathroom area, including the toilet paper (which was an interesting discovery).

I packed all of my stuff from my previous place and carried it over. Instant noodles are the quintessential student food of our generation.

The view out the window isn't particularly spectacular, but sometimes I can see kids playing in the alley below. I stopped watching them after they discovered me leering down at them one day. I guess I'm not really a creepy old man, but there's no point in practising.

Maybe I should buy a periscope.

And one of the more comical things I found was this glow-in-the-dark exit sign above my door. Even in pure blindness, it would be difficult to not find your way out of the room.

The area I'm in, which is named after the university's subway stop, has a more suburban feel to it. Conveniences are everywhere and I don't have to walk far to find what I'm looking for.

On the weekend I found a Vietnamese beef noodle shop. These are popular in Korea, and the taste is fairly good. But in Australia, there's a large Vietnamese community, so the beef noodles back home are excellent.

Back to the lab again. These days I'm pulling 8:40am until midnight as hours on occasion, but there are others here that do it more regularly. I always was a workaholic, so it doesn't bother me much. I enjoy the freedom that I have in organising my own timetable.
The machine in the photo above is called an autoclave. Because we need most of our equipment sterile before we use it, most things are autoclaved regularly. This machine applies high pressure, steam and heat to whatever is put inside. The result is that no bacteria can survive, and even viruses are broken down. Many people don't know, but bacteria and viruses are about as different as fish and rocks. Well, maybe not fish and rocks. More like fish and water.
You can see that the lid of the machine kind of looks like a submarine hatch. That's because this kind of seal is the best design for high pressure environments.

And if you autoclave things the wrong way, this is what happens. In this photo, the plastic lids of the test tubes melted and fused onto the tubes themselves, as well as the rack. We had to throw these away.

In the last blog post, I told you how we can get foreign DNA into bacteria by simply giving them a heat shock. Another way to do it is with this machine in the photo. It's called an electroporator and what it does is apply a short zap of electricity to your bacteria. What you do is plop the bacteria into a special tube with metal sides and mix them with the DNA you're interested in. The electrical charge pierces the cells and carries the DNA in with it, because DNA is negatively charged. But it all happens so quickly that the holes in the bacteria seal up fast enough that some of them can survive.

Here's our newest Ph.D student, Keonwoo, helping me to prepare the electrocompetent cells. Keonwoo is a funny guy and we get along well. He smokes a lot of cigarettes and his voice sounds a little like Smegol, but he's very chilled. His English name is Keanu, due to the similar spelling, but I pointed out that this was because the spelling was wrong. According to the revised romanisation of Korean, his name should be spelled Geonwoo. So I sometimes call him Geanu Reeves.

The weather has been warming up nicely, but the other day we had precipitation that was a mix between snow and rain. I'm sure there's a proper name for it, but I called it Snain. When spring fully arrives, I hope to start jogging again.

That's all from me! See you next time.

Life in the Lab

I had my first dream about an experiment last night. These days I'm dreaming (or at least remembering my dreams) more frequently than a few months ago. I heard as an explanation of dreams somewhere once, that during your dreams, your brain is busy sifting through the day's events and working out which memories should be permanently stored. The dreams we experience are a sort of conscious interpretation of the television-static-like jargon that is generated during this time. Anyway, I guess the fact that I dreamt about an experiment just goes to show that I'm probably thinking about them too much.

While I'd like to blog more about life in Korea in general, being in the lab for 75+ hours per week tends to limit the variety of photographs I have in my camera when it's blogging time. I hope that Lee's Korea Blog can still manage to be of interest to a wider audience as it inevitably makes a transition to something more like a 'Ph.D in Korea-Blog'.

But I must say that I am interested in what I'm studying. I guess my biggest challenge now is trying to stimulate your interest in what I'm up to these days. In the photo above is a petri dish with different colonies of blue and white bacteria growing on it. The black text just details the names of the chemicals that we're growing the bacteria on, and the red text is talking about the type of DNA that the bacteria are holding. The white bacteria are the ones that we're interested in, whereas the blue spots are the naughty ones that didn't do what we wanted them to. Bacteria are commonly used to store DNA and copy it for us, kind of like a photocopier. We even refer to them as 'libraries' at times. Such blatant exploitation of simple organisms to meet our own needs is probably worthy of more scholarly debate. But no one seems to care about bacteria these days.

Except when we're sick.

Laboratories all over the world often use very similar equipment, and there's a whole underworld of suppliers that specifically target the laboratory market. You never hear about these companies normally, because they only focus on advertising to their specific customers. In this photo, along the bottom line of the sticker above our fumehood, are some examples of the names of the suppliers that I'm talking about.
I guess that because most of the companies are started up by retiring professors, the names rarely get more catchy than 'Lonza' or 'Mupid'.

This is where we get our water from in the lab. It's a fairly souped-up filtration system that takes out whatever it is that makes tap water dirty. Because chemical reactions are often sensitive to things like heavy metals or chlorine, we need to be careful about the kind of water we use.
Did you know that pure water actually freezes at -42 degrees celsius? Zero degrees is when impure water freezes, and the ice crystals grow by forming around bubbles or dissolved impurities in the water. Most of the ice that we're acquainted with comes from water that isn't completely pure. But if you take out all of the impurities and try to freeze it, the water has a more difficult time forming ice, because it doesn't have anything to form crystals around. So to achieve the same effect, you need a lower temperature.

This is a PCR machine. PCR means Polymerase Chain Reaction, and it created a small revolution in the science world when it was invented. It basically uses a combination of special chemicals and temperatures to amplify DNA so much that you can see it and take a photo of it. This is important because DNA is usually invisible to us, so it's very helpful when we can see what we're working with. I'll show you a photograph sometime later.

One thing I have noticed is that they seem to be a little more lax about safety here. In Australia, you're not allowed to eat in the lab. But in Korea, they have coffee machines in operation a few metres away from the fumehood. I guess what doesn't kill you only makes you stronger.

This is a rather nifty device called a Speedy-Vac. What it does is dry things out very quickly. When you work with things dissolved in liquids a lot, you sometimes need to get the liquid out so that you can work with the stuff left over. It would seem logical to just heat it and get the water out, right? But the problem is that if you heat DNA too much, it disintegrates, which (as with most recently disintegrated things) tends to make it considerably less useful to us.
So what this machine does is spin things around like a Hill's Hoist in a vacuum environment. It sucks out the air in a kind of pressure chamber, which makes things evaporate without heat. The stuff you need doesn't get sucked out, because it's getting spun around so fast that inertia will keep it in place. Cool hey?

And here is my weapon of choice for most of the week. This is a pipette, and what it does is deliver precise amounts of liquid into whatever I'm making. It's important to know exact volumes when you're dealing with biochemicals, and this little thing is accurate to one-one thousandth of a millilitre.

Remember those tobacco plants growing in the lab that I showed you a few weeks ago? Well they've just started to grow flowers, so celebrations are in order. I had never seen a tobacco flower before, and I must say they're a lot prettier than I imagined. When I think about it, I probably would have imagined them to be sticky, black, cancerous things. But these are quite pleasant.
Economically important crops such as tobacco tend to have a lot more research funding directed at them. This means that we know a lot about tobacco, rice and corn, but not so much about Sturt's Desert Pea. We're not researching tobacco at all, but we use it in our experiments because there is a lot of information freely available for it. What we can then do is use tobacco as an analogy to other systems. Tobacco has big leaves, which makes it useful for things like microscopy or to track a bacterial infection.

What we're studying is rice, which I feel better about. It's nice to think of your research as eventually being beneficial to people who need it. Our rice in the greenhouse is starting to form grains.
Did you know that one rice plant, coming from a single seed, can yield around 10,000 grains of rice? It's amazing what a single plant can do with a bit of dirt and sunshine.

Science itself is not particularly difficult. The ideas can be complicated, but if you break things down and use less jargon, nearly everyone would understand the core concepts. The rest is just details.
To that end, I'm going to take you through the basics of a common thing we do called Bacterial Transformation. Transformation means getting some DNA (dissolved in liquid) inserted into bacteria so that they will do things with it for you. Bacteria can do all sorts of interesting things with the right kind of DNA. The first thing you do is take out your bacteria from the -80 degree celsius freezer. They live here in suspended animation when not in use.

Then you thaw them out slowly in an ice box. They're in the two circular tubes in the photo. You add your foreign DNA directly into the tubes, so the bacteria are floating around in solution.

The timer is used to keep a track of the time. Funnily enough.

Then what you do is plunge them quickly into a 42 degree celsius water bath. We call this a heat shock. For us humans, going from 0 degrees to 42 degrees all of a sudden isn't particularly interesting. But for bacteria it's another story. The 'skin' of most bacteria is actually more like an oily bubble that surrounds their inner fluids. When you change the temperature quickly, what happens is that their skins will form temporary holes as the heat stretches them rapidly. The holes eventually close up, but during this time, some of the DNA that was floating around in the liquid will enter the cell. That's how we get the DNA from the outside of the cell into the bacteria, creating a biologically modified organism. Pretty simple really.
You'd think that if it was so simple, a lot of drastic genetic modification would probably happen in natural situations.

Well, it does.

Then after their traumatic ordeal, we give the bacteria a nice shot of this stuff called Luria-Bertani broth. It's a mixture of everything that bacteria find delicious, and they basically throw a party if you give them enough. Bacterial parties are good, because they have a lot of babies and do interesting things with the DNA that we quietly slipped them before the party.

After all that science talk, you may be feeling like this. In this photo, Hoon and Eun-Hae are having a quiet nap on a Saturday in the lab. Rather than being a sign of laziness, it actually represents how much work they got done during the week.

With the weather warming up nicely, I went back to the frozen lake to have a look. It's all melted now and I tried to look for some of the fish that spend the winter under the ice.

I couldn't see any because the water was rather murky.

I'll occasionally eat out on the weekends when I'm by myself. There's not much difference between food in Seoul and Busan, but Busan has a lot more guk-bab (soup-rice). Around the corner from my place though, I recently found this dish, called kong-guk (bean soup) which I had never tried before. It's a brothy soybean stew that has cabbage and a bit of beef in it. It was fine, but nothing to go out of your way for.

Heather went to Australia this week and came back on Thursday. Because she's a good worker, the school lets her do things like that. Having not returned to Australia since arriving in 2006, I had a small shopping list for her. In the photo above are Mi-Goreng noodles and Extra chewing gum. You can't get these particular noodles in Korea, and I ate them a lot in Australia. As soon as Heather pulled them out, I cooked some up and a whole lot of memories came flooding back. Heather said they taste like Korean noodles.

I'd say they do about as much as Vegemite tastes like kim-chi.

We went out to the Gangnam area of Seoul, which is usually fairly busy. I hadn't been out this way since I moved. Seoul doesn't really have a city centre, it has hot spots of activity, of which Gangnam is one. There are a few more on the other side of the river.

This is the Kyobo building, which is the headquarters of a bookstore company. Korea has been investing heavily in education, so publishers and bookstores like this one have flourished. The country is striving for a knowledge-based economy, with electronics, robotics and biotechnology being focal points.

This guy was playing drums outside for a bar. There's always a lot of competition for exposure in Seoul, and there's only so much that a flashing neon sign will do for your business. Playing drums like this will certainly get you noticed, but whether that translates into more customers is questionable. The tune sounded something like ba-dum dum ching, ba-dum ba-dum ching!

It wasn't bad.

Here's Heather standing outside the Hard Rock Cafe in Itaewon. I had never been to a Hard Rock Cafe before, so it was interesting. All I knew about them was that they sell shirts and they're in a lot of big cities around the world.

I was right about the first two points, but I also found out that they have a lot of guitars hanging around the place.

I liked the decor and the atmosphere was classy but relaxed. When we walked in, they were playing 'You Shook Me All Night Long' by AC/DC. They're an Australian band who first got together around 30 years ago in a pub called the Pooraka Hotel in Adelaide.

The drinks here weren't especially pricey and we came back after meeting with Steve for dinner. Steve is an American adoptee who I met on the 2005 OKF tour, and is currently studying for his law degree in Tokyo. We were busy drinking and talking, so I forgot to get a photo of him. Steve reads this blog from time to time.

Hi Steve!

And to finish up with this week, here is a slippery dip on the side of a building here in Seoul. With land being so expensive, playgrounds are more of a luxury that can be easily outbidded by people with money. At least they're still trying.

That's all from me this time! I hope this blog can remain interesting and I'll continue to update for as long as I have things to talk about.