Yesterday morning was very stimulating - conversation with and seminar by Francisco Ubeda, a visiting theoretician whose focus on the evolution of intra-genomic conflict led him to a very nice model of the evolution of recombination hotspots. The seminar's audience was great too. I'm going to try to make a stop-motion animation of his model, but first I have to model eukaryotic chromosome replication, then mitosis, then meiosis, then crossing over, then initiation of crossing-over by double-strand break repair, then the role of hotspots in initiation, and then the hotspot conversion paradox. At that point I can make a model that incorporates a trans-acting sequence-specific modifier of hotspot activity. This may take some time....
But yesterday afternoon was very frustrating. I had brought my new batch of DNA-coated polystyrene beads to the optical tweezers apparatus, in the hope of testing whether competent cells would bind to them. But I never got to try this, because it was so difficult to get the tweezers to hold on to a bead. Almost every bead that got close to the laser focus was drawn in to it and then immediately spit out again (probably drawn in one side and out the other (the beads appeared to pop through the trap, rather than sticking at its focus). I halfway remember my biophysicist colleague telling me that the bead should approach the focus point from the front side, but I had no way of telling whether an out-of-focus bead was in front of the focus or behind it.
First I tried a chamber with B. subtilis cells and beads, then a chamber with beads but no cells. The beads were sufficiently sparse that finding ones to try to trap was inefficient, so I concentrated my bead stock and filled a fresh chamber. This only resulted in lots more beads popping through the trap.
The plane of focus is both the focus of the visible light that illuminates the image and of the laser that traps the bead. I'd been advised that trapping worked best when this plane was about 5 µ behind the coverslip surface (the top of the chamber), so I tried to maintain this position. It wasn't always at exactly the same setting on the micrometer that controls the focus position, because of minor variations in the thickness of the parafilm sheets that form the sides of the chamber. When I didn't have cells attached to the coverslip, I could still check this position once I had trapped a bead, by bringing the focus forward to a position where the coverslip pushed the bead back out of the trap (the laser focus point).
But even with the focus perfectly positioned, only very few beads stayed in the trap for even a few seconds. My colleague suggested trying 3 µ beads (mine were 2.1 µ) as she's had consistent success with them. But I couldn't get them to work much better than mine. Eventually I gave up. I think it may be time to set this whole project aside until we find a graduate student to take it on.
1 day ago in Variety of Life