Out of Place E. coli

[Charles Kopec]

I have been here one year and a half but I feel I wasted 8 months doing an experiment which produced beautiful negative result after negative result. A lot of people in the lab say I have been cursed by the ghost of Barbara McClintock because it turns out that the construct that I was given to by a former post doc in the lab was a bit flawed. I made all my mutations out of this construct and I was testing them all. Every week at our lab meeting I would come and say, look I did this mutation but it came out negative. We were always looking for the mutation that would yield us a positive result and we were always coming up with these hand-raising arguments to rationalize why it failed. We would say, well, this one failed because it did this or that to the shape of the protein or this one is really close to this other protein so the reaction site probably got mucked up. It turns out that the parent vector that I was given had an e. coli trasposon inserted into it, so what it ended up doing was it introduced a stop codon and truncated my protein and all of my mutations were doing nothing. They were pointless. I was always expressing this little fragment that was truncated and giving me my negative results. So everyone says that somehow I must have upset Barbara McClintock and she came down and made a transposon jump into my plasmid.
Well the protein I was working on is huge by standards of what we work on in the lab so I did not outright sequence the entire thing. What I usually did was sequence over the region of my mutation to make sure my mutation was fine but eventually I just sat down and sequenced the entire plasmid to see what the hell was wrong with the thing and eventually I found this little transposon! Actually, my protein was up on BLAST and my plasmid was up on BLAST so I would compare the sequencing results to make sure there were no point mutations, so all of the sudden I blasted it and I get back e. coli mobile element so I thought oh, let me check which sequence run this is. But I look and the transposon is smack in the middle of my protein! Yeah, I had one of those insane laughs because I realized 8 months of work was a waste of my life. But I have given up on that project and I have switched gears. But subsequently, I asked around wondering how often this happens and apparently it is rare, the kind of rare that makes me feel I should have bought a lottery ticket.

But the worst thing is the way the protein is made, it was located before the transposon. So when I would put my construct into the cells it would still glow because it would have the GFP but the GFP instead of being attached to my full protein was attached to a little non-functional tail. You see, you would never know that by looking at it. I think Barbara McClintock did not like the fact that I switched from corn, she must have wanted me to keep working on plants, but I switched.

Eventually, I actually met the post doc who made the construct and put it in the freezer and I wanted to just whack him over the head with my lab notebook or something for having put a transposon in his construct. It obviously was not his fault, oh, all grad students say that stuff inevitably happens. It is just one of those life lessons that hopefully I can appreciate a bit more after I have written and defended my thesis.