Split mCherry

I had a product that could have benefitted from using split mCherry to serve an AND function. Put the split mCherry in my usual mCherry spot in the recombination vector (2A’d with Puromycin, also), and I couldn’t see any visible fluorescence when both the small and big fragments were in the same cell. After some time, I saw a paper using split super-folder mCherry fused with the Spycatcher system, so I used that and that seemed to allow us to now see a shift in fluorescence off the background. I found another paper using an improved split super-folder mCherry (sfmCherry3C), and tested that, which seemed to work slightly better.

So while not perfect, in that there isn’t *complete* separation from the background distribution, it’s shifted away enough that it should serve my purposes (for now). Though, well, I’ll probably keep playing around with this (perhaps adding something like a leucine zipper?) and seeing if that helps increase fluorescence.

Dark culture media with RUBY

I like playing around with various recombinant DNA tools to see how they work and figure out if they’ll do something useful for me. Sometimes it works out amazingly, like iCasp9, which is a fantastic negative selection transgene. Other times, it’s not so clearly a success…

I recently ordered RUBY from Addgene (originally used by the depositing lab to darken plant roots) to see if I could turn my cultured cells visibly darker. I had actually messed around a little with this concept previously using tyrosinase, where it worked in making the cells darker, albeit one could only see the darker color either as a centrifuged cell pellet, or perhaps as a large overgrowing “colony” on an originally sparse culture plate. Well, I shuttled RUBY into my recombination vector and selected for cells expressing it. I don’t even think I saw dark cells this time (though I didn’t look very closely, since this is just a fun side-experiment), but I did notice that these cells had much darker media than their recombined siblings, like the cells expressing fluorescent proteins in the two wells to the right of it. So I’m not exactly sure what’s happening, but the chromogenic small molecule is definitely making it out into the culture media.

I guess I’ll just file this observation for now and see if it ever comes in useful at some point the future! hahaha.

Update: FYI, RUBY is *BIG*. Like ~ 4kb kind of big, since it encodes multiple enzymes within a chemical pathway. So def not a small chromoprotein kind of thing.

HEK293Ts with melanin

I think synthetic biology is really cool, and I like playing around with recombinant DNA elements so I can see how well they work in my own hands. If they work OK, then I just let that knowledge stew in the back of my brain until I can eventually figure out a use for it. Reading this paper by Martin Fussenegger made me realize just how easy it is to make cultured cells express melanin. Here was my first foray in creating melanin in HEK cells by overexpressing tyrosinase/

Cells pelleted in the tubes on the left are expressing tyrosinase. The cells pelleted in the tubes on the right are not.

Doesn’t quite work well enough to use as a general reporter (it’s really hard to tell in a cell monolayer, and only becomes noticeable as colonies of cells or in a pellet, like above), but still kind of fun to see. Let’s see if I find an eventual use for this in some future work.