The SUMO protease Ulp1 is an important regulator of the cell cycle in the model system Saccharomyces cerevisiae. When the cell cycle goes awry in humans, diseased states such as cancer and chromosomal abnormalities can result. Accordingly, a firmer understanding of how cell cycle regulators like Ulp1 work would further our knowledge of how these diseases come to be, and perhaps enlighten our ability to treat them. With this in mind, I set out to find out how Ulp1 is targeted to its substrates. Specifically, I cloned discrete regions of the gene coding for Ulp1 into overexpression plasmids, which I then used to create large amounts of purified, truncated protein for use in in vitro reactions.
Well, I’m officially no longer a fan of sticky end cloning. Granted, there is good news: I got sequences back for 2 more clones, but my cloning from earlier in the week failed. Miserably. Neither of the catalytic domain constructs produced bacterial colonies. Occasionally, there are technical reasons these things don’t work, such as if the protein product is toxic to the bacteria one uses to spin up copies of the plasmid. However, this isn’t the case for my protein, because it’s produced recombinantly by a biotech.
So after weeks of trying to get my other cloning reactions to work (two out of five are stalled at the moment), I finally bit the bullet and decided to actually try to sort out my sequencing data from my first two putative clones of the first region of my protein.
As this is a blog partially meant to educate its readers on the process of summer research, I thought I’d write on an integral part of the biological laboratory experience. As bacteria and other living organisms don’t, as a rule, conform to timetables set by researchers, it becomes necessary to come in to the lab on the weekends to set up your work for the coming week. For example, today, I came in to prepare media for a protein induction tomorrow, and to inoculate liquid cultures so I can have some DNA to work with tomorrow. Even with the most meticulous planning, these little visits can only be minimized with respect to their frequency and duration, never eliminated. When your work is progressing quickly, often it’s difficult to stay out of the lab, but when things stagnate for no apparent reason, the Sunday matinee becomes a far more appealing prospect…
…is currently in Dr. Forsyth’s 15 degree heat block! Today’s work involves a transformation of the final, catalytically dead active site into bacteria, and then confirmation work can begin on the clones I’ve created. With luck, I can identify putative clones to send off for DNA sequencing by monday, and begin overexpressions next week! Since so much of molecular biology involves waiting (an overnight wait here, three hours there), to maximize my time, I’ve had a side project or two involving SUMO-targeted ubiquitin ligases, which are moving quite quickly. In other news, keeping an organized lab notebook is a more frustrating endeavor than even the most intractable experiment.