This morning I went to the university across town to consult with a couple of experts about components of my CIHR proposal. My first meeting was with the physicist whose optical tweezers apparatus expertise I plan to rely on. Here are some issues we discussed:
To get evidence for USS-dependent binding, we should collect data for a histogram of rupture forces (i.e. how strong a pull is needed to break the bead free of the cell), for DNAs with and without USS. If binding is distinct from uptake, this distribution should be bimodal, probably with a much higher peak for the low 'binding' rupture force. She said this would be quite a lot of work (weeks, at least).
Can we stick H. influenzae cells onto a glass coverslip, (as the B. subtilis people did) rather than restraining them with a micropipette? This would make the manipulations much easier. She recommended trying BSA-coated coverslips as well as silane-coated ones. The coating will also help reduce non-specific sticking of DNA to the glass.
To test whether uptake is due to 'diffusive motor' or a 'power stroke' (I'll need to read up on these concepts) we should create a force-velocity curve. If we find that, below a critical stalling point, the velocity of DNA uptake is independent of the opposing force pulling the bead back into the center of the laser trap, we would conclude that uptake was due to machinery exerting a power stroke, whereas if we observed a gradual slowing of uptake with increasing opposing force, it would be due to a diffusive motor. I think Tfp use a power stroke, and a process driven by flow of ions (maybe the PMF (= proton motive force?) would be diffusive. The B. subtilis rate is largely independent of the opposing force on the bead, although it appears to somehow be generated by the PMF.
If uptake is indeed mediated by a short pseudopilus that acts as a ratchet on the DNA, we might be able to see the ratcheting in a velocity vs time (or force vs time?) graph. This would depend on the resolution of the velocity (or force?) measurements. If the pseudopilus is only, say 20 nm long (a guess of the thickness of the periplasm), we would need to detect fluctuations on this scale.
In principle optical traps can exert forces greater than 100 pN on trapped beads. However DNA deforms at 65 pN, allowing it to stretch. Not only will such stretching complicate length/velocity measurements but the deformation may perturb interactions with the uptake proteins, causing them to lose their grip on the DNA. So we should keep our measurements below about 60 pN.
She recommended making a flow chart to lay out the tests we will do and how the results will affect our hypotheses and guide our next steps.
murE mutagenesis planning
9 hours ago in RRResearch