Trials and tribulations of January / February research
1. Mammalian expression
2. E.coli expression
Unfortunately, both of them were blocked.
1. I’ve never done mammalian expression, so was having to read a huge amount to figure it out and getting nowhere.
2. My plasmids were mammalian, so I have to clone the insert into an E.coli vector, and I haven’t “officially” ever done cloning as a grad student. So trying to figure that out was also taking forever.
Then, I had a minor stress attack when Khalid called me from Germany to answer some of my emails he hadn’t gotten around to answering. And I suddenly realized, I was winding myself up and self-destructing and had to cut it out. Khalid also – who hadn’t known I was so stressed – suggested a different tack – to make a different RNA substrate and try it out on the enzyme I already had. That way I could work on something I knew how to do while also figuring out the new stuff. And it turns out – THAT has made all the difference.
I got my new substrate in finally this past Monday, and my enzyme is unbelievable. My student Sam has nicknamed it “Clark.” It went from 10% activity in an overnight reaction on our old substrate to 100% activity in under an hour on this new one. It also has multi-turnover capacities better than anything I’ve seen reported in literature, even at 4x less enzyme concentration than substrate. It’s incredible! Sam and I were blown away. The results look fake, they are so good. Usually, I have the opposite problem – results being bad. I’ve never had the problem of results looking too good to be true! What does one do in that scenario? LOTS OF CONTROLS. And repeats of the experiments.
Moving forward at light speed!
My enzyme activity has been a MAJOR bottleneck and was the locus of trouble at my second year report. Having this problem removed is a HUGE leap forward! Actually, this problem (aside from maybe 1-2 others I can see down the road) was really my biggest fear about my project. Now that this works, I have high confidence that the whole project can work once the bugs are worked out. It’s tremendously exciting!
So, we’re going to be working like a blizzard next week, both Sam and I, trying to characterize our enzyme on the new substrate and get splicing substrates ordered first thing. Khalid let me order a second gel electrophoresis instrument so we can run more gels at a time. I can’t wait to see what we’ll find! Light speed here we come! The sky has finally cleared! Look for my other posts today to see what other things have been happening in lab. :D