Future Ben

It’s true. I am now a master of science. I am looking at the results of my qualifying exam and it indubitably says, “pass.” Actually the oral exam was eerily smooth. There were a few conversational questions by my board, but other than that they just listened quitely, deliberated for 5 minutes, then brought me back in and said, “congratulations.” I thought I was going to sweat it out for 3 hours under a barrage of impossible questions. I was done in an hour and a half!

I feel strangely disapointed. With an impending sense that the other shoe is going to drop in the next few months. Transgenic mice are notoriously frustrating. But I must tiumph, for the future.

This week in lab I am presenting for lab meeting. Basicly, I ramble on about my weak conclusions based on weak data. That’s a little harsh, but nothing breaks down my confidence like having to tell somebody what I am up to. Especialy when they know more on the subject than I do. I am talking about MRI of course. When you are standing in a darkened room talking to a screen you can almost sense that your advisor really wants to correct you, but doesn’t want to embarass you in front of your lab mates. All in all it went pretty well.

What you are seeing is an array of bacterial cultures after being exposed to Manganese. The second top row is Mn in solution. What I am getting from this image is that Mn in a cell interacts with water much differently than when in a pure solution. There is a lot more to this image than just that, but that’s the gist of it. Next I will be putting my Mn binder through the paces and seeing how it compares to control cells.

This week in lab I am STILL working on constructs. I managed to get my genes into expression vectors and get tags on them, mostly. I still want to put flourescent reporters on the same mRNA though.

I have added an IRES eGFP to nRamp2, my metal pumping protein. Now, whenever it is expressed you will also see GFP as an instant reporter on its activity. It is important to note that this tells you nothing about whether nRamp2 has been folded and sent to the membrane like it is supposed to. For that I need to use the tag I added.

That leaves Ferritin M. I can’t very well use the same color reporter, so I have to make something new. The Tsien lab at UCSD has come up with a few decent red fluorescent proteins. mCherry is the one I will be using.

In order to make an IRES mCherry I am using pBS, a vector meant just for cloning. First I am putting in the IRES through blunt end ligation then I’m adding mCherry by its BamH1 EcoR1 sticky ends. Once its assembled then I will put it into the expression vector pCDNA FerritinM-HA that I made last week.

Meanwhile my Manganese binding protein project is moving along swimmingly. I am expressing it in bacteria right now. The great thing about this protein I have discovered is that it sucks up trace amounts of Mn from the media and gives excellent contrast against a control.

Its funny how I spent all of last semester getting these tags and cloning out all of these iron proteins only to have this Mn binder to fall into my lap and work like a charm. I hate science!

Whats going on this week? Right now I’m trying to create a menu of expression vectors containing an epitope tag with an IRES eGFP. What this means is that whatever gene I express using one of these vectors will have a special tag on one end or the other plus eGFP will show up wherever the gene does.
This is great for detection purposes because not only do the cells expressing my gene of interest fluoresce green, but I can probe for my tags as well. I wish they just sold these things online. I’ll post maps of these constructs, and if anybody wants to save themselves weeks worth of molecular biology, just send me a self addressed stamped envelope and I will send you an aliquat of any of my DNA. Oh. Good idea. I am going to start a construct gallery this weekend.