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Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature
In This Article
Summary
The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.
Abstract
We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4–8 µm/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.
Introduction
Active matter is differentiated from conventional passive matter due to its capability to convert chemical energy into mechanical work. A material that possesses such capability can consist of living or non-living entities such as bacteria, insects, colloids, grains, and cytoskeletal filaments1,2,3,4,5,6,7,8,9,10. These material entiti....
Protocol
1. Preparation of MTs
CAUTION: In this step we purify tubulins from bovine brain tissue. Bovine brain may cause variant Creutzfeldt-Jakob disease (vCJD)35. Therefore, the brain waste and related solutions, bottles, and pipette tips should be collected in a biowaste bag and disposed of as biohazardous waste according to the rules of the institution.
- Purify tubulins from bovine brain (modified from Castoldi et al.36).
- Transp.......
Representative Results
Preparing the kinesin-driven, MT-based active fluids requires both kinesin and MTs. The MTs were polymerized from labeled tubulins (steps 1.3 and 1.4) that were purified from bovine brains (step 1.1, Figure 2A), followed by recycling to enhance purity (step 1.2, Figure 2B). The kinesin motor proteins were expressed in and purified from E. coli (steps 2.1 and 2.2, Figure 2B).......
Discussion
Controlling active matter in situ opens the door to directed self-organization of active matter4,5,24,28,54. In this article, we present a protocol for using temperature to control kinesin-driven, MT-based active fluids in situ, based on the Arrhenius characteristic of the system29,30,
Acknowledgements
Plasmid K401-BCCP-H6 was a gift from Dr. Zvonimir Dogic. This research was supported by Dr. Kun-Ta Wu's start-up fund in Worcester Polytechnic Institute. We thank Dr. Zvonimir Dogic for the protocols to purify and label tubulin and to synthesize active fluids. We are grateful to Dr. Marc Ridilla for his expertise in protein expression and purification. We thank Dr. William Benjamin Roger for assisting us with building the temperature-controlled stage. We acknowledge Brandeis MRSEC (NSF-MRSEC-1420382) for use of the Biological Materials Facility (BMF). We acknowledge the Royal Society of Chemistry for adapting the figures from Bate et al. on Soft Matter
Materials
| Name | Company | Catalog Number | Comments |
| (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid | Sigma-Aldrich | 238813 | Trolox |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| 3-(Trimethoxysilyl)propyl methacrylate, 98%, ACROS Organics | Fisher Scientific | AC216550050 | |
| 3.2mm I.D. Tygon Tubing R-3603 | HACH | 2074038 | Water tubes |
| 31.75 mm diameter uncoated, sapphire window | Edmund Optics | 43-637 | Sapphire disc |
| 3M 1181 Copper Tape - 1/2 IN Width X 18 YD Length - 2.6 MIL Total Thickness - 27551 | R.S. HUGHES | 054007-27551 | Copper tape |
| Acetic Acid | Sigma-Aldrich | A6283 | |
| Acrylamide Solution (40%/Electrophoresis), Fisher BioReagents | Fisher Scientific | BP1402-1 | |
| Adenosine 5'-triphosphate dipotassium salt hydrate | Sigma-Aldrich | A8937 | ATP |
| Alexa Fluor 647 NHS Ester (Succinimidyl Ester) | Thermo Fisher Scientific | A20006 | Far-red fluorescent dye. Alexa 647 can be pre suspended in dimethylsulfoxide (DMSO) before mixing with microtubules (1.3.3.2.) |
| Amicon Ultra-4 Centrifugal Filter Unit | Sigma-Aldrich | UFC801024 | Centrifugal filter tube. Cutoff molecular weight: 10 kDa |
| Ammonium Persulfate, 100g, MP Biomedicals | Fisher Scientific | ICN802829 | APS |
| Ampicillin Sodium Salt (Crystalline Powder), Fisher BioReagents | Fisher Scientific | BP1760 | Ampicillin |
| Antivibration Table | Nikon | 63-7590S | |
| Avanti J-E Centrifuge | Beckman Coulter | 369001 | |
| Bacto Agar Soldifying Agent, BD Diagnostics | VWR | 90000-760 | Agar |
| Biotin | Alfa Aesar | A14207 | |
| Bucket-plastic white - 2 gallon | Bon | 84-715 | Water bucket |
| Calcium Chloride | Sigma-Aldrich | 746495 | CaCl2 |
| Catalase from bovine liver | Sigma-Aldrich | C40 | |
| CFI Plan Apo Lambda 4x Obj | Nikon | MRD00045 | 4x air objective |
| C-FLLL-FOV GFP HC HC HISN ero Shift | Nikon | 96372 | GFP filter cube |
| CH-109-1.4-1.5 | TE Technology | CH-109-1.4-1.5 | Thermoelectric Cooler (TEC) |
| Chloramphenicol, 98%, ACROS Organics | Fisher Scientific | C0378 | |
| Cooling block | N/A | N/A | Custom milled aluminum |
| Coomassie Brilliant Blue R-250 #1610400 | Bio-Rad | 1610400 | Triphenylmethane dye |
| D-(+)-Glucose | Sigma-Aldrich | G7528 | |
| Dimethyl Sulfoxide (Certified ACS), Fisher Chemical | Fisher Scientific | D128 | DMSO |
| DL-1,4-Dithiothreitol, 99%, for biochemistry, ACROS Organics | Fisher Scientific | AC165680050 | DTT |
| DOWSIL 340 Heat Sink Compound | Dow | 1446622 | Thermal paste |
| ETHYL ALCOHOL, 200 PROOF ACS/USP/NF GRADE 5 GALLON POLY CUBE | Pharmco by Greenfield Global | 111000200CB05 | Ethanol |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid | Sigma-Aldrich | E3889 | EGTA |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | 798681 | EDTA |
| Fisher BioReagents Microbiology Media Additives: Tryptone | Fisher Scientific | BP1421 | Tryptone |
| Fisher BioReagents Microbiology Media Additives: Yeast Extract | Fisher Scientific | BP1422 | Yeast extract |
| Fluoresbrite YG Microspheres, Calibration Grade 3.00 µm | Polysciences | 18861 | Tracer particles |
| Glucose Oxidase from Aspergillus niger | Sigma-Aldrich | G2133 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| GpCpp | Jena Bioscience | NU-405L | Guanosine-5'[(α,β)-methyleno]triphosphate (GMPCPP) |
| GS Power's 18 Gauge (True American Wire Ga), 100 feet, 99.9% Stranded Oxygen Free Copper OFC, Red/Black 2 Conductor Bonded Zip Cord Power/Speaker Electrical Cable for Car, Audio, Home Theater | Amazon | B07428NBCW | Copper wire |
| Guanosine 5'-triphosphate sodium salt hydrate | Sigma-Aldrich | G8877 | GTP |
| Hellmanex III | Sigma-Aldrich | Z805939 | Detergent |
| HEPES Sodium Salt (White Powder), Fisher BioReagents | Fisher Scientific | BP410 | NaHEPES |
| High performance blender machine | AIMORES | AS-UP1250 | Blender |
| His GraviTrap | GE Healthcare | 11003399 | Gravity Column |
| Imidazole | Sigma-Aldrich | I5513 | |
| IPTG | Sigma-Aldrich | I6758 | Isopropyl β-D-1-thiogalactopyranoside |
| Isopropyl Alcohol 99% | Pharmco by Greenfield Global | 231000099 | Isopropanol |
| JA-10 rotor | Beckman Coulter | 369687 | |
| L-Glutamic acid potassium salt monohydrate | Sigma-Aldrich | G1501 | K-Glutamate |
| Lysozyme from chicken egg white | Sigma-Aldrich | L6876 | |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M2670 | MgCl2•6H2O |
| MES sodium salt | Sigma-Aldrich | M5057 | 2-(N-Morpholino)ethanesulfonic acid sodium salt |
| MOPS | Sigma-Aldrich | M1254 | 3-(N-Morpholino)propanesulfonic acid |
| MP-3022 | TE Technology | MP-3022 | Thermocouple |
| N,N,N',N'-Tetramethylethylenediamine 99%, ACROS Organics | Fisher Scientific | AC138450500 | TEMED |
| Nanodrop 2000c UV-VIS Spectrophotometer | Thermo Fisher Scientific | E112352 | Spectrometer |
| Nikon Ti2-E Nikon Inverted Microscope | Nikon | MEA54000 | |
| Norland Optical Adhesive 81 | Norland Products | NOA81 | UV glue |
| Novex Sharp Pre-stained Protein Standard | Thermo Fisher Scientific | LC5800 | Protein standard ladder |
| NuPAGE 4-12% Bis-Tris Protein Gels, 1.5 mm, 10-well | Thermo Fisher Scientific | NP0335BOX | SDS gel |
| Optima L-90K Ultracentrifuge | Beckman Coulter | 365672 | |
| Parafilm PM996 Wrap , 4" Wide; 125 Ft/Roll | Cole-Parmer | EW-06720-40 | Wax film |
| Pe 300 ultra Illumination System Single Band , 3mm Light Guide control Pod power supply | Nikon | PE-300-UT-L-SB-40 | Cool LED Illuminator |
| Phenylmethanesulfonyl fluoride | Sigma-Aldrich | 78830 | PMSF |
| Phosphoenolpyruvic acid monopotassium salt, 99% | BeanTown Chemical | 129745 | PEP |
| Pierce Coomassie (Bradford) Protein Assay Kit | Thermo Fisher Scientific | 23200 | |
| Pierce Protease Inhibitor Mini Tablets | Thermo Fisher Scientific | A32953 | |
| PIPES | Sigma-Aldrich | P6757 | 1,4-Piperazinediethanesulfonic acid |
| Pluronic F-127 | Sigma-Aldrich | P2443 | |
| Poly(ethylene glycol) | Sigma-Aldrich | 81300 | PEG. Average molecular weight 20,000 Da |
| Potassium Hydroxide (Pellets/Certified ACS), Fisher Chemical | Fisher Scientific | P250-500 | KOH |
| PowerEase 300W Power Supply (115 VAC) | ThermoFisher Scientific | PS0300 | DC power supply of the gel box |
| PS-12-8.4A | TE Technology | PS-12-8.4A | DC power supply of the temperature controller |
| Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle | Sigma-Aldrich | P-0294 | PK/LDH |
| Quiet One Lifegard Fountain Pump, 296-Gallon Per Hour | Amazon | B005JWA612 | Fish tank pump |
| Rosetta 2(DE3)pLysS Competent Cells - Novagen | Millipore Sigma | 71403 | Competent cells |
| Sharp Microwave ZSMC0912BS Sharp 900W Countertop Microwave Oven, 0.9 Cubic Foot, Stainless Steel | Amazon | B01MT6JZMR | Microwave for boiling the water |
| Sodium Chloride (Crystalline/Certified ACS), Fisher Chemical | Fisher Scientific | S271-500 | NaCl |
| Sodium dodecyl sulfate | Sigma-Aldrich | L3771 | SDS |
| Sodium phosphate monobasic | Sigma-Aldrich | S8282 | NaH2PO4 |
| Streptavidin Protein | Thermo Fisher Scientific | 21122 | |
| Sucrose | Sigma-Aldrich | S7903 | |
| TC-720 | TE Technology | TC-720 | Temperature controller |
| Tris Base, Molecular Biology Grade - CAS 77-86-1 - Calbiochem | Sigma-Aldrich | 648310 | Tris-HCL |
| Type 45 Ti rotor | Beckman Coulter | 339160 | |
| Type 70 Ti rotor | Beckman Coulter | 337922 | |
| Type 70.1 Ti rotor | Beckman Coulter | 342184 | |
| VWR General-Purpose Laboratory Labeling Tape | VWR | 89097-916 | Paper tapes |
| VWR Micro Cover Glasses, Square, No. 1 1/2 | VWR | 48366-227 | Glass coverslips |
| VWR Plain and Frosted Micro Slides, Premium | VWR | 75799-268 | Glass slides |
| XCell SureLock Mini-Cell | ThermoFisher Scientific | EI0001 | Gel box |
| ZYLA 5.5 USB3.0 Camera | Nikon | ZYLA5.5-USB3 | Monochrome CCD camera |
References
- Wioland, H., Lushi, E., Goldstein, R. E. Directed Collective Motion of Bacteria under Channel Confinement. New Journal of Physics. 18 (7), 075002 (2016).
- Wioland, H., Woodhouse, F. G., Dunkel, J., Kessler, J. O., Goldstein, R. E.
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