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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Engineering

Styring af strømningshastigheder for 3D-aktive væsker med mikrotubulus ved hjælp af temperatur

Published: November 26, 2019
doi:
10.3791/60484
, , ,
1Department of Physics,Worcester Polytechnic Institute, 2Department of Physics,Brandeis University

Summary

Målet med denne protokol er at bruge temperatur til at styre strømningshastigheden af tredimensionelle aktive væsker. Fordelen ved denne metode ikke kun giver mulighed for regulering af strømningshastigheder in situ, men også giver mulighed for dynamisk kontrol, såsom periodisk tuning flow hastigheder op og ned.

Abstract

Vi præsenterer en metode til at bruge temperatur til at tune strømningshastigheder af kinesin-drevne, microtubule-baserede tredimensionale (3D) aktive væsker. Denne metode giver mulighed for tuning hastigheder in situ uden at det er nødvendigt at fremstille nye prøver for at nå forskellige ønskede hastigheder. Desuden giver denne metode mulighed for dynamisk kontrol af hastighed. Cykling temperaturen fører væsker til at flyde hurtigt og langsomt, periodisk. Denne styrbarhed er baseret på Arrhenius-karakteristikken af kinesin-microtubule-reaktionen, der viser et kontrolleret gennemsnitligt strømningshastighedsområde på 4 – 8 μm/s. Den præsenterede metode vil åbne døren til udformningen af mikrofluidisk enheder, hvor strømningshastigheder i kanalen er lokalt tunable uden behov for en ventil.

Introduction

Aktivstof er differentieret fra konventionelle passive stoffer på grund af sin evne til at omdanne kemisk energi til mekanisk arbejde. Et materiale, der besidder en sådan kapacitet kan bestå af levende eller ikke-levende enheder såsom bakterier, insekter, kolloider, kerner, og cytoskeletale filamenter1,2,3,4,5,6,7,8,9,10. Disse materielle enheder interagerer med deres naboer. I større målestok, de selv-organisere sig i enten turbulente-lignende hvirvler (aktiv turbulens) eller materialestrømme11,12,13,14,15,16,17,18,19,20. En forståelse af selvorganisering af aktivstof har ført til forskellige anvendelser i molekylære Shuttles, optiske enheder, og parallel beregning21,22,23. At bringe ansøgninger til det næste niveau kræver kontrol ud over selvorganisering. For eksempel, Palacci et al. udviklet en hematite-indkapslet kolloid, at selvkørende kun når de udsættes for manuelt kontrolleret blåt lys, hvilket førte til fremkomsten af levende krystaller24. Morin et al. etablerede kontrol af rullende Quincke kolloider ved hjælp af en tunable ekstern elektrisk felt, hvilket resulterer i kolloid strømmer i en væddeløbsbane-lignende kanal25. Disse tidligere værker demonstrere rollen som lokal kontrol i applikationer og fremme videnbasen af aktive stof.

I denne artikel fokuserer vi på styrbarhed af kinesin-drevne, mikrotubulus (MT)-baserede 3D-aktive væsker. Væskerne består af tre hovedkomponenter: MTs, kinesin Molecular Motors og depletants. De depletanter fremkalde en udtynding kraft til at bundte MTs, som senere bridged af motor klynger. Disse motorer går langs MTstoward plus enden. Når et par bridged MTsis antiparallel, de tilsvarende motorer gå i modsatte retninger. Men motorerne er bundet i en klynge og er ude af stand til at gå fra hinanden, så de i samarbejde glider fra hinanden par af MTs (interfilament glidende, figur 1a). Disse glidende dynamik ophobes, forårsager bundter af MTsto forlænge indtil nå deres Buckling ustabilitet punkt og pause (extensile bundter, figur 1B)26. De brudte bundter er udglødet af nedbrydningen kraft, som efterfølgende strækker sig igen, og dynamikken gentage. Under processen med den gentagne dynamik, røre bundt bevægelser de nærliggende flydende, inducerende strømme, der kan visualiseres ved doping med micron-Scale røbestoffer (figur 1C). Sanchez et al. og henkin et al. har karakteriseret de gennemsnitlige hastigheder af røbestoffer, konstatering af, at hastigheder var tunable ved at variere koncentrationerne af adenosin trifosfat (ATP), depletants, motor klynger, og MTs19,27. Men, en sådan tunbarhed eksisterede kun før aktiv væske syntese. Efter syntese, den tunbarhed var tabt, og væsker selvorganiserede på deres egen måde. For at kontrollere aktiv væske aktivitet efter syntese, Ross.et al. rapporteret en metode ved hjælp af lys-aktiveret denne af motor proteiner, så væske aktivitet, der skal tunet til og fra ved hjælp af lys28. Mens lys kontrol er praktisk i form af lokalt aktivering af væsker, metoden kræver redesigne strukturerne af motor proteiner, sammen med at ændre de optiske stier i et mikroskop. Her giver vi en brugervenlig metode til lokal styring af væske strømme uden mikroskop modifikation, samtidig med at motor strukturen bevares intakt.

Vores metode til lokal tuning aktiv fluid flow er baseret på Arrhenius loven, fordi kinesin-MT reaktion er blevet rapporteret at stige med temperatur29,30,31,32. Vores tidligere undersøgelser viste, at temperatur afhængigheden af den gennemsnitlige hastighed af en aktiv fluid flow fulgte Arrhenius ligningen: v = a exp (-EA/rt), hvor a er en præ-eksponentiel faktor, R er gaskonstanten, EA er aktiveringsenergien, og T er systemets temperatur33. Derfor er flydende aktivitet følsom over for temperatur miljøet, og system temperaturen skal være konsistent for at stabilisere motorens ydeevne, og dermed væskestrømmen hastighed34. I denne artikel viser vi brugen af motorens temperaturafhængighed til løbende at finjustere strømningshastigheden af aktive væsker ved at justere system temperaturen. Vi demonstrerer også forberedelsen af en aktiv væskeprøve, efterfulgt af montering af prøven på et mikroskop stadie, hvis temperatur styres via computer software. En forøgelse af temperaturen fra 16 °C til 36 °C øger den gennemsnitlige strømningshastighed fra 4 til 8 μm/s. Desuden er tunbarhed reversibel: gentagne gange øger og sænker temperaturen sekventielt accelererer og decelererer flowet. Den demonstrerede metode gælder for en lang række systemer, hvor de vigtigste reaktioner adlyder Arrhenius-loven, såsom MT gliding assay29,30,31,32.

Protocol

1. forberedelse af MTs Forsigtig: i dette trin renser vi tubuliner fra bovin hjernevæv. Bovin hjernen kan forårsage variant Creutzfeldt-Jakob sygdom (vCJD)35. Derfor bør hjerne affald og relaterede opløsninger, flasker og pipettespidser opsamles i en bioaffald taske og bortskaffes som biologisk farligt affald i henhold til institutionens regler. Purify tubuliner fra kvæg hjernen (modificeret fra Castoldi et al.36). Transpor…

Representative Results

Klargøring af kinesin-drevne, MT-baserede aktive væsker kræver både kinesin og MTs. MTs blev polymeriseret fra mærkede tubuliner (trin 1,3 og 1,4), der blev renset fra kvæg hjerner (trin 1,1, figur 2A), efterfulgt af genanvendelse for at forbedre renhed (trin 1,2, figur 2B). Kinesinmotorproteinerne blev udtrykt i og renset fra E. coli (trin 2,1 og 2,2, figur 2B)<sup c…

Discussion

Kontrol af aktiv substans in situ åbner døren til styret selvorganisering af aktivstof4,5,24,28,54. I denne artikel præsenterer vi en protokol for brug af temperatur til at kontrollere kinesin-drevne, MT-baserede aktive væsker in situ, baseret på Arrhenius-karakteristisk for systemet29,30,</s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Plasmid K401-BCCP-H6 var en gave fra Dr. Zvonimir Dogic. Denne forskning blev støttet af Dr. kun-ta Wus opstartsfond i Worcester Polytechnic Institute. Vi takker Dr. Zvonimir Dogic for protokollerne til at rense og mærke Tubulin og til at syntetisere aktive væsker. Vi er taknemmelige for Dr. Marc Ridilla for hans ekspertise i protein ekspression og rensning. Vi takker Dr. William Benjamin Roger for at hjælpe os med at bygge det temperaturstyrede stadie. Vi anerkender Brandeis MRSEC (NSF-MRSEC-1420382) for brug af den biologiske materiale facilitet (BMF). Vi anerkender det kongelige kemi selskab for at tilpasse tallene fra BATE et al. på Soft Matter33.

Materials

(±)-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
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MicrotubuleActive FluidTemperature ControlKinesinATPPolyethylene GlycolPhoto-bleachingTracer ParticlesPolyacrylamideGlass Slide
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Bate, T. E., Jarvis, E. J., Varney, M. E., Wu, K. Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature. J. Vis. Exp. (153), e60484, doi:10.3791/60484 (2019).
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