Biomod/2011/Columbia/MotorProTeam:ProjectDevelopment
Brainstorming for the project began in February 2011. The following ideas were taken into consideration for the competition:
- Genetic engineering of hemoglobin
- Microtubule sheets for force multiplication of kinesin
- Electric power from ATP
- Smart indicator supermolecule (multi-receptor recognition by multifunctional drug delivery vehicle)
- Slow protein unfolding by motor proteisn
- Self-Healing
- Nanofactory using molecular shuttles
- Circuit assembly by molecular shuttles
We ultimately decided on an adaptation of the second idea (microtubule sheets for force multiplication of kinesins). This choice was based on interest, feasibility, and the potential application of the prospective projects. We aim to create a universal transporter that can selectively carry nano- and micro- scale cargo over millimeter distances. This procedure took place in a flow cell.
To harvest the microtubules, 0.5 μL MgCl2, 0.5μL GTP, 0.6μL DMSO, and 10.9 μL BRB80 were mixed in a small tube. 6.25μL of this growth solution was added to a 20 μg aliquot of rhodamine labeled tubulin. The aliquot was vortexed and heated for 30 min at 37°C.
MT100 was made using 490μL BRB80+5μL Taxol+5μL of the prepared microtubules.
BRB80CS was prepared using 58.54μL BRB80 and 1.46μL Casein
the motility solution was prepared using 81.6μL BRB80+2.43μL Casein+1μL (Taxol [TX], DG [D-Glucose], Catalase [Cat], Dithiotheritol [DTT], ATP)+10μL MT100
The antifade solution was prepared with 81.6μL BRB80+2.43μL Casein+1μL (Taxol [TX], DG [D-Glucose], Catalase [Cat], Dithiotheritol [DTT], ATP)
Kinesin motor solution (for a density of 2,000/μm^2) was prepared with 40μL BRB80+0.6μL ATP+1.2μL CS, and two minutes later, 7.44μL Kinesin.
To make the flow cell, double sided tape (or vacuum grease) was added to each side of a cover slip. A small coverslip was added ontop. This creates a (insert size here) volume for the solutions to flow. A pipet was used to flow solution in from one side, and filter paper collected the solution from the other.
20 μL BRB80CS solution was flowed into the cell. Five minutes later, 30 μL kinesin motor solution was flowed. Five minutes later, 30μL motility solution was flowed. 5 minutes later, 30μL antifade solution was flowed. Then, the flow cell was imaged under the microscope.
Describe the current method here. Describe how ours is better.
From this point, we met to design the structure of the device.
Initially, we aimed to create a raft-like surface, that is, an array of microtubules that were of one length, pointed in the same direction, and were connected either by biotin-streptavidin bonds. This idea, although elegant, was unfeasible. When multiple microtubules are connected, they form spool-like structures rather than a raft-like structure. Controlling the length of each microtubule also presented a challenge to the idea. We considered using dynein as an alternate to kinesin because of its stiffness. But regardless, the nature of microtubules, when put together, would make this structure impossible.
One potential alternate was a cylindrical system that operated on bead-geometry as opposed to the motility assay. A cylinder would be constructed of microtubules, and cargo would attach to the cylinder. Kinesin molecules on a fixed surface would pass the microtubules along thereby moving the cargo. The benefit of this structure is in the high surface area to volume ratio of the cylinder. This would allow a large amount of cargo to be carried along. Unfortunately, however, the bead geometry is much less effective than the gliding geometry. We therefore continued theorizing and discussing potential structures for the universal transporter.
The next, and probably most promising idea resembled a nano-truck constructed from microtubules, motor proteins, and a sheet of PDMS as the loading dock. Essentially, we would have a sheet of PDMS (which we could then geometrically manipulate to maximize carrying efficiency) with an oxidized, hydrophilic bottom surface. Microtubules would attach to the bottom surface. Extending the metaphor, these microtubules are the analog wheels of the transport vehicle.
Images of possible structure ideas:
First, one sheet of PDMS without any geometric modification would serve as the test structure. This allows us to prevent any steric complications from affecting the results.
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Next, we would work with a topless box-like structure. This allows more space for the particles which we plan on transporting to attach.
(insert image here)
If the topless box works, a surface with a grated pattern (think teeth with gaps between) would be the ideal structure. We can create this by building a mold into which the PDMS is poured.
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The structure would have to be small enough to fit inside a flow cell. In order to achieve this type of sizing, silicone wafers were patterned with a photoresist mask. Handling such small particles manually would be virtually impossible, so a combination of microtransfer molding and a lift-off technique would be used to acquire small squares of PDMS.
PDMS can be spin-coated onto the surface of the mask in order to achieve a thin layer. After letting the PDMS cure, it
After deciding on a structure, we investigated methods to align microtubules. The simplest method appeared to be using magnetic fields to align the microtubules. The procedure was unfeasible to perform within the constraints of the lab because magnetic fields had to be applied in the process of growing the microtubules. This posed a road block (no pun intended).
One alternative was using force within the flow cell to align microtubules. AMP-PNP was used to replace ATP in the solutions, and two antifade solutions were prepared (ATP antifade was flowed first, followed by AMP-PNP antifade). A large amount of ATP antifade was prepared, and it was flowed at a rate of 5 to 6 μL per second. Allegedly, this shear flow would align the microtubules, and if applied for a long enough duration (the specific timings are still unclear), the microtubules would eventually align by polarity. In our experiments, however, the microtubules did not align.
A more promising method to align microtubules entailed polymerizing microtubules uniquely from one end and then cross-linking them to the surface. Microtubules polymerize faster on the positive end than on the other. It is thus possible to use this polarity to control their growth. This procedure entails growing the microtubules inside the flow cell. A flow cell was prepared with half of the flor area covered with tape, and casein was flowed through. The tape was removed, and kinesin was flowed through, followed by an antifade solution. The kinesin does not stick to the previously tape-covered portion because there was no casein sticking to that portion. Microtubules were flowed through the cell, and would therefore be forced to line up along the barrier of the kinesin region to the blank region. (insert rest of procedure here).
