Transitioning from a life of woe to a life of wow: from malaria to yeast

When I started the blog not very long ago, the intention was to post an article once a week. But last week came and went without posting anything. I can’t even really blame this on my Birthday and having my family in town as this all happened after the self-imposed deadline of Thursday. I have some articles in the works but find all the legwork of diving into the primary literature unappealing at this moment. So instead I will discuss my personal experience with transitioning from biochemistry to yeast genetics. After years struggling with the malaria parasite Plasmodium falciparum in my PhD; cloning from a 70% AT-rich genome, trying to express, purify, and crystallize proteins that are generally larger and more disordered than their human homologues in E. coli, and trying to do western blots with not very good antibodies, I thought I would do myself a favor and focus on the model eukaryotic organism, Saccharomyces cerevisiae. I would still study the biological processes I was interested in, autophagy and protein trafficking, but in a system with some proper tools. Plus yeast smells a heck of a lot better than bacteria!

So how are things going nearly 3 months in?

One thing that has taken getting used to is the amount of planning that is required when working with yeast. I was very used to deciding to purify a protein from bacteria and two days later, having the materials to do so. Once the proteins were purified and stably frozen away, experiments could be conducted as soon as I planed them. But with yeast, more time is required to first grow on a plate from a glycerol stock, then transform a plasmid and wait three more days for colonies, then start a small overnight culture, and the next day: microscopy. A week can easily go by where I don’t really have anything tangible to show. This is thankfully changing, but requires a lot of multi-tasking and planning ahead so that while strains are growing for one project, I am doing experiments for another. Right now, my method of juggling this has evolved into each day reviewing the previous days “to-do” list and writing a new one for that day. I can only imagine there are or will soon be, lab apps that will send text reminders for different experiments.

I am also learning that yeast are tricky little guys. They readily undergo homologous recombination, which is great for manipulating the genome, but they will do anything to stay alive and seem to shuffle things around as need be so that I am left wondering where the heck in the genome did my GFP tag go? I cannot complain too much about this, as I have deleted a gene in less than a month, compared to the oft-predicted one-year time frame for P. falciparum. I was hoping the microscopy would be easier with yeast, but really they are not much bigger than the asexual forms of Plasmodium (5-10 µm vs 1-2 µM). I still find myself looking longingly at the images of human cells or tissues appearing on the microscope computer screen next to me. But at least tagging yeast genes is easy, allowing for live microscopy. However, tagging a gene can be deleterious for that protein’s function and I have struggled for weeks trying to C-terminally tag several proteins in a complex without success. So I am about to begin the laborious task (this is relative, of course) of N-terminal tagging several genes to hopefully get around this problem. Which is to say that science is never easy.

So how are things going? Generally pretty good. I am learning a lot about genetics and working with yeast. Maybe in 3 more months I will actually have some worthwhile results…or be working for a brewery.