1/24/05
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!