If I do an experiment that uses 50 nm magnetic beads, am I using nanotechnology? If the experiment involves a new use for these beads, am I developing nanotechnology? More generally, is all molecular biology nanotechnology?
Anyway, what am I going to do with these beads? I want to find out whether the USS polarizes the direction of DNA uptake. That is, when the USS sequence interacts with the DNA uptake machinery on the cell surface, does only the DNA on one side of the canonical orientation get pulled into the cell, at least until the end has been brought in?
My latest model of the mechanism of uptake initiation predicts that at the initiation step a type 4 pseudopilus pulls in the DNA on the right side of the USS core, while the core is bound tightly to a receptor protein on the surface (this could be the secretin pore-forming protein). If the core remains tightly bound, uptake could continue only on this side until the end of the fragment is reached. This would require that the force exerted by the pseudopilus on the DNA be less that that needed to pull the USS core free from its receptor. Bringing in the end of the DNA would allow transport across the cytoplasmic membrane, which would reverse the direction of uptake. Under this version of the model, this reversal of direction and the stronger pull exerted on the DNA by the cytoplasmic membrane machinery would detach the USS core from the receptor and bring in the DNA on the left side.
This model of uptake is much more detailed than the available evidence supports. I see this as a strength, not a weakness. The model makes many very specific and testable predictions, and at this stage of investigation it's more important for a hypothesis to be testable than to be correct. Maybe that's true at any stage.
I still like the idea of using laser tweezers to investigate the polarity question, but my physicist collaborator points out that this single-molecule method may lack the resolution we need to answer the question. Tweezers are best at measuring forces, not movement of cells.
So I'm back to thinking about blocking the ends with beads. Our previous attempts used what we now realize were giant beads (streptavidin-agarose), far bigger than the cells and much too big and porous for the task. This improved approach will use beads that are only 50 nm across, which should be plenty big enough to prevent uptake as the secretin pore is thought to be only 6-7 nm across at its widest. They can be purchased with streptavidin bound to their surface, making it easy to attach them to DNA just as we did with the agarose beads.
Our original experiments labeled one end of a short USS-containing fragment with 32P and blocked the other end with a (giant) bead. We predicted that the orientation of the the USS with respect to the bead would determine whether the 32P got inside the cell, but found that orientation had little effect. I think we should repeat these experiments using the 50 nm magnetic beads, paying careful attention to the kinetics. The original experiments used 220 bp fragments; because such short fragments might behave differently than long ones, we should also repeat the analysis with fragments that are one or a few kb long (easy - we just use the whole USS plasmid rather than its 220bp insert).
Maybe we can then combine use of the beads with the tweezer analysis, examining forces rather than distance changes as originally planned. If the tweezer analysis of the force acting on the DNA shows that the inner membrane machinery exerts a stronger force than the pseudopilus, then blocking the right end of a long fragment with a bead (left end held in the laser trap) should allow weak-force uptake but prevent the transition to strong-force uptake. Flipping the orientation of the USS should. . . . . no, the transition would still be blocked, this time by the large trapped polystyrene bead at the other end. The flipped USS orientation would affect movement of the cell rather than the kind of force.
Friday fabulous flower - alotta hosta
6 hours ago in The Phytophactor