White knuckle ride...

I always knew that a PhD would be a rollercoaster but now it's turning into such a white knuckle ride, I'm not sure I would ever have got on in the first place had I only known.

Science is full of ups and downs - that just the nature of it. You can spend months, even years  doggedly following a line of enquiry, only for it to end up fruitless or flawed. Or a whole day carefully setting up an experiment only for the critical machine to 'have an off day'. Working on living things, of course, adds a whole new dimension in which things go wrong. Problems are inevitable...but then you have those moments when a result throws up something completely unexpected, and sends your research off done an exciting, unforeseen new direction. Suddenly all the frustrations don't seem to matter.

Right now I could certainly do with some more ups. As I wrote in my last blog post, time really isn't on my side if I am going to get enough good data for a PhD. So as soon as the new year started, I began carefully setting up a HUGE gene expression assay, to try to work out what happens on the molecular level when my parasite of study, Striga gesnerioides, infects its host. I had over 120 plates of Arabidopsis seed squashed into my cabinet which took hours and hours to prepare but then - disaster! Almost every plate was contaminated with bacteria. The seedlings, now useless,  had to be binned. A whole month of work wasted. I really can't afford setbacks like this - a couple more could tip the balance in terms of whether I have any chance of finishing in time. 

Very poorly looking Arabidopsis seedlings! 

Fortunately, I have identified the probable cause. It seems that the tap for distilled water that I suspended the seed in after sterilising them,  is not as clean as it should be - when I put some water on an agar plate just to see what happened, sure enough bacterial colonies appeared. So from now on, I sterilise my water in the autoclave just to be sure. I have also tightened up any over potential entry points for nasties - using ultra sterile pipettes tips; irradiating my pipettes with UV light, autoclaving the Eppendorf tubes, even taking my watch off (surely a treasure trove of germs!). So far the next lot of (120 plus) plates seem free from contamination, so fingers crossed!

Then I had my second disaster, one which nearly made me made me walk out of the lab for good. One of my most interesting results so far has been with a certain Arabidopsis mutant that fails to produce a particular protein. For some reason it is much more susceptible to Striga, so I propagated the seed to give me enough to do more experiments to investigate it further. This requires care, especially if you have other plants in the same cabinet, to make sure that they don't cross fertilise each other and create hybrid offspring. But I was confident that I had used the special 'aracons ' ( plastic tubes used to keep  flowering Arabidopsis plants separate) correctly.  When I tested the protein expression in these plants however - disaster!!! The plants were still producing the protein! Somehow, the plants must have mixed themselves up and the mutation had been lost. I was devastated and spent a very low evening wondering what in earth I was going to do now for the rest of my PhD. 

Our distilled water tap - was this the source of the bacterial contamination? 

But then I had another look at the data - and it turns out that the protein exists in two different forms and my plants were actually one of these, but not another. When I checked the position of the genetic mutation, this made perfect sense : it should disrupt the disrupted the coding sequence of just one of the protein forms, but not the other. And in fact, given that these different forms have very different functions, it actually makes a really interesting result, which could set the direction of the rest of my experiments.... it certainly interested my supervisors anyway!

Lots of new plates of Arabidopsis seed crammed into my growth cabinet - so far, bacteria free!

So a typical week in science, traversing the whole range from despair and misery, to a minor breakthrough. Last week I also had the pleasure of interviewing an old friend for a careers feature I am working on for a magazine. Having completed a PhD investigating drought resistance in Sorghum, she decided to follow her passion for science communication and now works as a medical writer. One of the things that struck me the most was how refreshing she found it to to have a role where your hard work would always be rewarded with a physical output, rather than being derailed by things outside your control. I couldn't agree more!