Full disclosure: no matter how hard you try, no matter how careful you are, things can, and most certainly will go wrong. I say this from first-hand experience. Allow me to explain. As I described in my previous post, I am working with chromobodies, which are tiny nanobodies fused to a fluorescent protein. To create our chromobodies we are using a new procedure known as gateway cloning. You can read up on the procedure in more detail here, but basically the process involves two steps in which the DNA sequence for our chromobodies is inserted first into one plasmid and then recombined into a second plasmid to create an expression clone. The expression clone is a plasmid containing a promoter that will cause the chromobody to be overexpressed within the cell. The idea is that once we transfect cells with our expression vector, they should begin producing chromobodies that will bind to their antigen targets (either actin, lamin, pcna, or dnmt1) within the cell. We then put the cells under the confocal microscope which excites the fluorescent proteins within our chromobodies allowing us to visualize the chromobodies bound to their targets within the cell. Here’s a time-lapse video of cells that have been transfected with a lamin chromobody containing GFP (green fluorescent protein):
I have finished cloning the DNA sequences for each of my four chromobodies into four different expression vectors (using the gateway cloning protocol). And I have also transfected HEK cells with my four different chromobodies.
So far so good right? Except things happen, things you can’t control. For example, you might get only three out of your four chromobodies to successfully transfect. This is what happened to me. Our chromobody for pcna would not transfect. We sequenced the DNA for our chromobody and low and behold discovered that it contained a mutation that caused the fluorescent protein to be dysfunctional (meaning that it did not emit light after being excited by the laser in the confocal). So what do you do? You go through the whole cloning procedure again and hope that the next time around you don’t get a mutation. Of course, as luck would have it, my second clone also had a mutation. It was a different mutation, but it resulted in the same loss of chromobody functionality. So right now we are currently on round three of cloning for our pcna chromobody.
Moral of the story: when life gives you a mutation, clone, clone again.
