User:Brian P. Josey/Notebook/2011/02/24
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A Slight Shift
Yesterday afternoon, I met with Koch to discuss my research, and come up with some ideas for my future research. We talked about the magnets that I've created so far, and the ferritin project in general. While it would be nice to take Gary's idea all the way to fruition, and put the ferritin inside of a cell and move proteins around, it would be very difficult to do before I graduate. Unfortunately, I don't really have time on my side for this project. I will graduate next May, and I am looking into getting an REU for this summer. Coupled with the fact that next spring semester will be mostly devoted to looking at graduate schools and writing my thesis, I have maybe six months of productive research time left. With the cells, learning how to properly conjugate ferritin to proteins and perform in vivo experiments would take up a lot of time. However, with all of the other productive research going on, Koch and I brainstormed a path that could be more productive.
With the optical tweezers now working, and Andy's thesis coming to a close, we have a lot of activity that is going on, and a lot of resources that I could tap into. With the FEMM models that I've created before, I have always struggled with a way to physically measure the forces to compare to my models. One idea was to measure Stoke's drag of a magnetic bead, and use that to calculate the force. But Koch also suggested something that is a little bit more involved, and would give me a chance to learn more than just magnetics. While we were talking about it, we wrote up everything on a whiteboard to keep it in track. Below is a picture of what we wrote up, and then below that is a step-by-step explanation of what it all means.
1. Polymerizing MT's with biotin tubulin We can create microtubules that have biotin tubulin in them, and is done carefully, we could also make it so that the microtubule grows out in only one direction. Koch actually began writing a paper on this while he was a postdoc at Sandia, the paper is here. One of the figures illustrates exactly what I mean.
If we do this, then we can ensure that any bead we attach to the microtubules remains is at one end, and any forces that we generate on it will be easy to simulate, and not give us some weird forces from the center of the microtubule. One of the key things to consider at this point is how would we know if we successfully connected the beads to the microtubules. I will try to answer this question, as well as gain the technical skills required to created microtubules.
2. "Shearing" the microtubules We would want the microtubules to be shorter than they are usually created for the next step. The length of the microtubules is approximately 20 μm long, but we would want them to be only ~1 to 5μm long. The easiest way to do this is to shear them in a syringe. Across the radius of a syringe needle, any liquid flowing through it has a laminar flow, but between different radii, there are shear forces between them due to the differing velocity of the liquid. By pulling in and ejecting the microtubules repetitively, we could cut them down to size. This step is fairly simple to do, all I need to do is find a resource that covers the procedure, Koch mentioned he has some, and follow up on it. It then gets a little more complicated when we try to find out if they are the proper length.
3. Stick to beads After we have cut the microtubules down to the proper size, we would then connect them to a plastic bead using the protein steptavidin. This protein would connect the biotin directly to the bead, and essentially cover the bead with a layer of microtubules that can interact with kinesin. I think of this step as being analogous to a meatball covered in short strands of spaghetti. The open question here is also, how would we know that we succeeded, and what kind of test can we perform to prove it?
4. Motility Assay Now that we have a bead covered in microtubules, we plan to flow them into a flow cell containing kinesin to see if they will interact with the kinesin. If we get a form of consistant movement, or any other noticeable pattern, this will be a great check of our experiment, and will allow us to move onward to the next, and most difficult step.
5. Motility with tweezers This is where things begin to get tough. Everything before will have their own challenges, but this one is the big guy. Here we would hold the beads with the optical tweezers so that it connects to the kinesin proteins, and locks into place. After carefully monitoring the bead's movement, we would find a point where it is staying in place with very little motion. At this point we would then shear it off and measure the force needed to do that. After doing this several times, we would ultimately get a good measure of the forces needed to disconnect the microtubule coated beads from the kinesin.
6. Introduce magnets After we have collected enough data from step 5, we would then be able to move on to using magnetic beads. We would coat the bead in microtubules, as explained above, and then flow it into a flow cell containing the kinesin and wait for it to get trapped by the kinesin, and then apply a magnetic field until it is sheared off. At this point, we then can measure the magnetic force needed to move the bead, and we can compare it to the calculated forces I generated in FEMM.