Reflections on Lab Work

I've just come to the end of my project for this year. Last week was the usual flurry of tidying, organising the freezer, and making sure all my stuff is on the Lab Computer so that anyone else who wants it can use it. I tried very hard to feel some sort of nostalgia about leaving, but to be honest all I managed was a sense of supreme exhaustion and slight relief. Besides, I'll probably be nipping in an out of there to collect results pictures, protocols, etc. for my write-up.

So I thought I'd just do a quick run-through, for anyone who's interested, about my lab experiences. I've done four lab projects to date, and enjoyed every one of them. I can't even say which one I enjoyed the most, they're all special in different ways.

1) My First Project Ever was working on bacteriophages (viruses that infect bacteria). It was fairly heavily supervised, I was working with a PhD student and helping her on her project. It was probably the most fun I've ever had in a lab; there was no pressure for a big write-up (just a small report for the funding body) or excessive pressure for me to get results, and I felt very useful being able to help. It was also the first time I'd been paid for lab work, and got that amazing rush of "woah....people will give me actual money for this!"

I started this blog during that project. The entries from back then make me laugh now. :)

2) Second project was for my course, where I switched to working on Streptomyces. Again, I was working quite closely with a PhD student, but she had kids and used to have to leave the lab about midday to take care of them. By the time the first week was up we'd worked out a system; in the morning we'd go over all the work I needed to get done, and in the afternoon she'd leave and I'd get on with it. It was quite a nice step up, as I was still being supervised, but I was doing the work all by myself.

The blog kind of died in that period, because it was insanely busy and I hadn't found out about research blogging yet.

3) The third project was the synthetic biology one, which wins the award for Most Stressful Project so far. As it was meant to be student-based we (there were seven of us, only four of whom were biological scientists) were pretty much dumped in the lab and left to get on with it. We had meetings with the supervisors once a week, and we had a PhD student to help us out (for which we were incredibly grateful) but other than that it was all down to us.

Considering I was the only one in the lab who'd ever done a project before it's amazing we managed anything really! I've got a lot of blog posts from that time, mostly because I spent about four weeks in the middle of it failing to make two point mutations. This involved lots of waiting around for gels to run, PCRs to happen, and ligations to fail, during which I would happily type away at my computer. Also at some point around then I discovered researchblogging.org and completely took off with the research side of things.

4) The project I'm currently writing up. This one has been the first project I've really thought of as my own (which makes the lack of conclusive results a bit harder to take). The lab is quite small, and as the PI and post-doc were quite busy last term, I was organising experiments and procedures pretty much on my own. I'm looking forward to the write-up, especially now I've drafted it and discovered that I actually do enough to write up. I feel more personally attached to this project than any one I've ever done before. The knowledge that my little samples are going to be sitting in the freezer for a long while before anyone bothers to dig them out actually does hurt a little. The next three weeks of write-up are going to be fun though :)

I'm quite surprised and proud that I've managed to keep up regular blogging throughout the course of the project.

5) I've already organised a summer project! As usual, there will probably be only the vaguest details of what I'm actually doing on the blog, especially as this project really should lead to a paper. But stay tuned for more bacteria-related posts. I'm sort of hooked on blogging now, and it'll probably take a lot to get me to stop.

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Yay Procrastination!

To reassure the people I know who read this blog, I am actually working quite hard. However I decided to take a bit of time out today to write out my own skippy's list for Lab Rat laboratory work. This is, in effect, the Other Dissertation, based around my project last term. All of them come from things done, or discussed by either me or my fellow student Lab Rat during the project:

Thing’s Lab Rat has learnt in the Lab

1) Bacteria are to be referred to as ‘bacteria’, ‘microbes’ or ‘bugs’ not ‘bloody-minded bastards’
2) Warning labels are to be taken seriously
3) The Bunsen burner is not ‘out to get you’
4) Neither is the autoclave
5) Flame sterilised items remain hot for a while after being taken out the flame
6) If you must sing, sing quietly
7) Stick figures are not allowed on reports
8) Neither are smiley faces or any kind of emoticon
9) The phrase “It’s OK, no one will notice” should NEVER be uttered
10) Your PI can tell the difference between bacteria and fungal contamination
11) The autoclave does not have a mind of its own, and should not be blamed for any contamination
12) Making Darth-Vader breathing sounds while working under the fume hood is not necessary
13) Bacteria are not to be described as evil, no matter how many rounds of mutation they’ve been through
14) Sliding down the corridor is not encouraged, especially when holding plates
15) Juggling of any laboratory equipment should not be attempted
16) There is no such thing as approximate accuracy
17) Repeating an experiment until it gives you the correct results is not proper scientific technique
18) The above rule always applies, no matter how close to the deadline you are
19) Your lab coat is not a trench coat
20) There is a limit to how much glassware can be balanced on top of a lab book
21) ‘Bench space’ should not be an oxymoron
22) COSHH stands for ‘control of substances hazardous to health’, attempts to pretend it stands for anything more obscene are in bad taste
23) Molten agar does not remove fingerprints
24) The UV light should not be used for tanning [just for the record, this was never tried, only half-jokingly discussed]
25) The bacteria are not your minions, and should not be referred to as such.
26) Maniacal laughter is not encouraged
27) Thanking God in three different languages is not necessary, no matter how wonderful your results turn out to be
28) The computers are to be used for analysing data, not reading webcomics
29) The above rule applies even if the webcomic in question mentions bacteria
30) Many things in the lab sometimes produce smells of burning. This is no cause for total panic
31) Neither plates, nor agar bottles have the capacity to move on their own
32) Lab reports need not be written in Iambic Pentameter
33) Mutant bacteria are given numbers, not ‘codenames’
34) X-23 is not a number
35) The words ‘gloopy’ ‘thingy’ ‘unfortunate’ ‘awesome’ and ‘combobulated’ should not be used in scientific reports
36) Plate is not a verb (as in ‘to plate’)
37) Bacteria are killed by autoclaving, not by repeated stabbing with a spreader, no matter how satisfying the latter may be
38) The difference between 10?g and 100?g is not ‘just another zero’
39) Laboratory antibiotics should not be used to heal personal illnesses
40) If the thought of doing something makes you giggle, that thing is probably prohibited within the laboratory

Number 36 came up surprisingly often in my dissertation. "The bacteria were plated onto an agar disk..." "Plate 30 microlitres of bacteria" etc. it just flows so naturally.

Anyway! Back to G proteins...

How bacteria make antibiotics

There are many different types of antibiotics bacteria can make, but my lab project (now finished, alas) was concentrating mostly on a type called polyketides. These are not just antibiotics, some polyketides can also be antifungals and anticancer agents too, so it's not surprising that quite a lot of work has been done characterising their formation.

Here is the molecular structure of erythromycin. Like many polyketides it is circular, which at first appears to be a bit of a headache to synthesise. The way it's put together, though, is actually very clever. The backbone of the circular section is made up first, using a system of modular enzymes that pass the growing chain along like a conveyor belt, adding new residues at each stage. Then the straight chain is curled up into a ring, and finally the two side residues (the ones on the bottom right of the chemical structure, that look a bit like squashed rectangles with dents in them) are stuck on.

So here is the picture that has appeared on every slide show in every lab meeting we've had this term, showing the formation of the straight-chain backbone before it gets curved into a circle:
Ignoring the little letters (which are just names of enzymes) it really does look a lot like a conveyor belt. At each stage the chain is lengthened, before finally being taken off and twisted around onto itself (to form a circular molecule called DEB). The modular nature of this system is fascinating to work with, but a real problem to sequence. DNA sequencing techniques work mainly by chopping the genome up, sequencing the bits, then trying to stick them back together and modular repeats tend to confuse them.

The last stage, going from DEB to erythromycin, is just a matter of decoration. Although the squashed-rectangle additions (glycosylases, added by glycosylation I believe) look complex, they are quite common molecules that get added onto things in the cell. Glycosylase residues and glycosylation enzymes are very common.
And that's how bacteria make polyketides :)

Scanner Woes

How many times have I heard it on deviantart.com? The continual cry of "Oh noes! The scanner ate my picture!" And now I'm making the same complaints about mine.

Actually, my plate pictures haven't been too bad. Although a couple of them are somewhat ... darker and fuzzier than they could be. As there are no labels for anyone to pinch my results with, I think I am justified on posting a few on here:

That one's alright. A bit dark though. At least the contrast can be seen. In case anyone was wondering, what you are seeing is bacteria streaked across the plate and left to grow (marked by the black line across the plate) and the another bacteria grown on top of it, which has subsequently been killed.

But as this may turn into a paper (oh please! *crosses fingers and hopes*) I can't be any more specific.



This one (on the right) hasn't really come out at all. meh. There's not much I can do except tell people what they should be seeing and hope it all works out alright. So what you should be seeing is a patch of dead overlay (the light bit) that follows the line of the mould (crosshatched black pen), but is not actually present under most of the mould.


I have a dissertation to write up based on these! *panic*

On the plus side, my scanner seems to have also created some results of its own. Take a look at this bioassay:

See the white lines around the circles on the first two rows? They were not at all obvious in the photo. Although when I squint at the photo now I can kind of see them.

Yes, that is my handwriting at the bottom of the photo. Which was taken by yours truly in the (very old) lightbox, narrowly avoiding getting an accidental blast of UV light as well (UV light is used to take pictures of gels, and nobody bothers to switch the switch back to 'white light' when they've finished; noticed just in time)

I am very proud of all my results :) Which has probably confirmed for a Certain Special Someone that they are indeed going out with a very nerdy little thing.

=D

Jumping DNA

Here's a bit of random information while I wait for my agar to melt...

Bacteria have a remarkable genome plasticity. They are able to mop up spare DNA in the environment, take pieces from circling bacteriophages (viruses that infect bacteria for the uninitiated) and exchange parts of their genome with bacteria from another species. The species boundary can be very wide as well, the bacterial equivalent of a mouse nicking bits of DNA from an elephant and incorporating it into it's genome.

In order to jump from one genome to the other (bacteriophage and other viruses) the DNA must be flanked by so called 'transposable elements' usually shortened to 'transposons' because molecular biologists are lazy when it comes to saying unnecessary words. (also, I suspect because 'transposons' sounds more scifi and scientists have a distressing tendency to be geeky like that). These transposons code for enzymes that cut the DNA out and paste it elsewhere, essentially allowing it to jump around between various genomes, being expressed and replicated in different bacteria.

ooop, there goes the sodding microwave. We have a new one after the old one stopped working (about three weeks after I entered the lab. PURE COINCIDENCE) and this one hits a pitch which is just slightly higher than the comfort level.

Incidentally, does anyone know how to do those fancy 'cut' things in blogs? Like when there's a blue underlined 'read more' label which whisks you away to the rest of the blog post. I'd really like to do that but I don't know how...

On feeling ill...

I suspect having a cold isn't fun whatever job you're in. Coughing and sneezing in public tend to make people look at you oddly, and try to discreetly shuffle away in case they catch it. In a lab however, it's a whole new level of awkward because everyone around you is working with little eppindorfs and petri-dishes and trying their hardest not to get them contaminated.

Contamination is the One Big Evil in microbial biochemistry. And I've essentially turned into a walking contamination spray. I am already worrying for the fate of the plates I poured today, and it's only a matter of time before people start following me around with ethanol'd paper towels and disinfect everything I've touched.

I'm even starting to want to do it. Thankfully most of my work is done under a laminar hood, which is like a large box with a glass pane in the front which separates me from the plates and the bacteria. Which means that if I cough at the ceiling my plates should manage alright.

If they get contaminated I will scream. This is already the third time I've done this procedure. The first time the actual antibiotic we were using was contaminated, so we just grew nice little colonies of black mould. The second time I forgot to dilute the mixture down, so I was pipetting tiny, tiny amounts of liquid out and all the results were screwy. The problem is, is that the bacteria take five days to grow, which means I've already wasted two weeks and have no results.

And I'm ill. Meh. Welcome to science, two steps forward, six steps backward. :(

Where does the disk go?

Part of what my supervisor is doing for her project involves growing bacteria then challenging them by putting little antibiotic disks on the plates and seeing if the bacteria die or not. She can't make it in today, so she asked me yesterday if I could put the disks on the plates for her.

The procedure was fairly simple and wouldn't take long, so I agreed. I came in this morning all bright and early, and suddenly realised I had no idea where on the plates I was supposed to put the disks.

It the middle? At the edge? Where the bacteria are growing?

It's my fault for acting so competent in my first week. It made her form the impression that I know what I'm doing. :(

Lab Rat in its Natural Habitat

I'm back in a lab! More excitingly, a different lab. I moved in today, so I've just about been allocated bench space and shown where the autoclave is. Today's tasks included cleaning said bench (I pretty much just doused it with70% ethanol and waited for the ethanol to evaporate, I do not want anything contaminating the bacteria I'll be working with) and mixing up a load of media so that I can put it above my bench where it can sit and look all pretty.

The Lab equivalent of hoovering and putting up the curtains basically. As soon as the autoclave has finished (the autoclave is basically just a big pressure cooker which sterilizes everything) my bench will have:

• Four durans (large bottles) of Tap Water Media for growing bacteria in
• Four flat-bottomed flasks of LB media for overlaying plates and growing different bacteria on (used to test if the first bacteria are producing antibiotics)
• One small pot of 40% glycerol (our bacteria occasionally produce spores, and these are stored in glycerol)
• Two pots of sterilized toothpicks (for picking out isolated colonies of bacteria)
• Three empty sterilized flat-bottomed flasks, for as yet undisclosed purposes
• One empty measuring cylinder with foil over the top
  
• Two boxes of pipette tips

And I've just remembered that it should probably have double distilled water as well. Bugger. I will ask my supervisor about that, but water is used quite often and it would probably be helpful to have some around. Although my bench is right next to the MiliQue machine (which occasionally produces noises like a drain with a bad stomach and in some mysterious way ionises the water. Way beyond my MEH unfortunately) so I might be able to get away with it.

These will be placed on the shelf above the Lab bench, apart from the pipette tips which will be on the bench itself, along with my Lab Notebook (yes, it does deserve the capital letter), a biro, a marker pen that is running out of ink (for labelling the bottles), a roll of autoclave tape that probably isn't mine, a little rack for holding eppindorf tubes (ooh, that reminds me, I need to get a rack for the bigger tubes) and a bunsen burner stand that I've yet to move onto someone else's bench.

I'm home =D