Appleyard et al - Vanderbilt University

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The mixture is run in a PCR machine with a primary denaturation cycle at 94 °C for 3 min. Thirty amplification cycles follow, running 1 min at 94 °C for melting, 1 min at 48 °C for annealing, and 7 min of elongation at 72 °C. Each amplification cycle approximately doubles the concentration of the desired 3500 bp segment. A final elongation cycle of 14 min at 72 °C completes the PCR amplification. The processed tethers are cleaned using a QuiaQuick purification kit Qiagen. The tether cloning protocol is also available in the supplementary information. 12 To form DNA tethers between the coverglass surface and a bead, a 1:1 mixture of 20 pM DNA complexes and 1 wt % 1 m streptavidin coated silica beads Bangs is made and incubated for 4 h at 4 °C. A flow cell is created as described previously. A 1:5 dilution of 20 mg/mL anti-digoxigenin Roche in PBS 100 mM phosphate buffer, pH 7.4, 0.138M NaCl, 0.0027M KCl, Sigma is made and further diluted in 1:10 PBT 100 mM phosphate buffer, pH 7.5, 0.1% Tween, Fisher Scientific. The flow cell is loaded with 25 L of the anti-digoxigenin solution and incubated for at least 40 min at room temperature. To prevent nonspecific attachment, 200 L of a 1 mg/mL dilution of casein Sigma in PBT is loaded into the flow cell, using a vacuum to draw the liquid through the flow chamber. The casein solution is incubated for 20 min at room temperature. The bead:DNA complexes are pelleted by spinning at 9000 g for 6 min and the supernatant is removed. The bead:DNA complexes are resuspended in PBT and diluted by a factor of 4. Then 25 L of bead:DNA complexes are loaded into the flow cell using the vacuum and incubated for 20 min. As a final wash step, 800 L of a 1 mg/mL dilution of casein in PBT, is drawn through the flow cell using the vacuum. This process produces DNA tethers between the etched glass coverslip and the silica beads. A complimentary description of this process can be found in Ref. 3. PCR production of the DNA tethers can be done far ahead of time, with DNA samples stored frozen at −20 °C. Preparation of the slides must be done immediately prior to use and is best done by an instructor who is familiar with the protocol. Once loaded on the microscope, visual examination of the slide allows the identification of tethered beads through their restricted Brownian motion. When located, the sample is moved to position the tether in the center of the trap. The height of the bead above the surface is adjusted so that the bead is just touching the surface. This adjustment can be accomplished by finding the point at which the bead begins to defocus when the coverslip is moved closer to the trap, and positioning the bead and trap just above this point. Picomotor based stage movement translates the tether 2500 nm out of the center of the trap, then begins stepping the bead back through the trap in 30 nm increments at an average rate of 100 nm/s. Bead position data are acquired simultaneously with the sweep. As the tether moves through the trap, the bead is captured by the optical trap, creating a situation where the tether is fully stretched, allowed to slacken, and then stretched again. The stretching curve initially obtained reflects the displacement of the bead out of the trap on both sides with a flat, slack region in the center versus the stage position Fig. 8. The stage position versus detector voltage data is converted to force versus stage position using calibration parameters Sec. III. One half of the tether extension is extracted from the data by truncating the curve from the midpoint of the plateau Fig. 8, label B to one end of the linear region of Fig. 8. Bead displacement x bead out of the center of the trap versus stage position x stage of a 3500 bp DNA tether. The cartoons illustrate the relative position of the trap, bead, and tether. The labels A and C indicate the approximate end of the linear portion of stretching curve. Label B is located at the center of the stretching curve. The flat area indicates a slack tether where the DNA is under no tension. the extension curve Fig. 8, label A or C. This segment of the curve yields data from a slack tether to the end of the linear region of force applied by the trap. The tether extension is calculated as follows, taking into account the bead radius r and the height h above the surface see Fig. 9, inset, x = h 2 + x stage − x bead 2 − r, where x is the tether length, x stage is the stage position, and x bead is the displacement of the bead out of the center of the trap. The values for p and c are extracted by performing a fit of the converted data to the interpolation of the wormlike chain, Eq. 7, using a nonlinear curve fitting algorithm such as nlinfit Matlab see Fig. 9. Alternatively, students can work backward to determine the position and stiffness calibrations by using the DNA stretching curve as an internal calibration standard. The calibration factors can be extracted by fixing p and c to calculate a force versus stage position curve and fitting the acquired data by optimizing the stiffness, k, and position calibration, . C. DNA tether stretching: Experimental results Accepted persistence lengths for dsDNA fall between 40 and 50 nm, and the contour length for a 3500 base pair strand is expected to be around 1180 nm. 29,30 The wormlike Fig. 9. Force versus extension curve for one of the 3500 bp DNA tethers. The wormlike chain fit gives p =29.9 nm and c =1077.8 nm. The inset cartoons show the relevant distances for calculating the tether extension x. 8 12 Am. J. Phys., Vol. 75, No. 1, January 2007 Appleyard et al. 12
form a construction set for students to build a simple microscope. Community contribution of further experiments is highly encouraged on the online resource. 12 Fig. 10. Rhodamine labeled microtubules excited with the 532 nm laser; the fluorescence emission peak is at 585 nm. The excitation zone is approximately 20 m in diameter. chain fit applied to a variety of our data sets produced persistence lengths between 10 and 50 nm with contour lengths from 1000 to 1200 nm. It takes a number of tries and practice to obtain a clean stretching curve. One slide contains many tethered beads to examine and five to ten stretching curves can be obtained in an hour. Improvements in centering the bead over the tether on the coverslip prior to an experiment and more accurately setting the height of the bead above the surface would help to further refine the results. A portion of the error in measuring p and c is due to the uncertainty of the calibration parameters. The instrument can assay force-extension curves for DNA, giving results close to accepted literature values culminating in an excellent single-molecule laboratory module. VI. EXPERIMENT: ADDITIONAL MODULES Further experimental development is encouraged to take advantage of the fluorescence and detection capabilities of the instrument. For example, rhodamine labeled microtubules can be visualized easily with this instrument as the peak excitation occurs at 535 nm and the emission peak is at 585 nm. Preparation of fluorescent microtubules like those in Fig. 10 is given in Ref. 12. These microtubules can be used as a base for a gliding filament assay, bead motility, or kinesin stepping module. 34 The persistence length of microtubules can be used by monitoring their thermal motion or through direct buckling using the optical trap. 32 The calibration module can be expanded by using solutions of different viscosities and varying the bead diameter to see how these parameters affect trap stiffness and the forces involved. The DNA stretching module can be extended using a higher power laser to demonstrate DNA shearing and unzipping or the opening of hairpins. With the flexibility of the optics, other basic microscopy modules can be developed. Basic techniques in optical design and measurement can be examined, such as measuring the beam diameter with a razorblade. 35 Advanced students can build and align the trap as part of a long term project; most labs will probably find assembly by students to be a large time commitment and prefer to use prebuilt traps. For courses that emphasize optics, components of this instrument the lamp, condenser objective, tube lens, and camera can VII. CONCLUSIONS An inexpensive, fully functional optical trap can be constructed for use as a teaching tool for undergraduate laboratory environments. The integrated position detection, stage movement, and fluorescence provide a solid foundation for many in-depth experiments. Laboratory modules geared toward understanding optical trapping fundamentals, exploring single molecule biophysics, and examining molecular motors have been created. The flexibility of this design promotes further experimental development. ACKNOWLEDGMENTS This work was supported through funding from the Biological Engineering Department at MIT. Laboratory advice from Dr. Peter So and Maxim Shusteff aided in the development and applications. We are grateful to Dr. Karen Fahrner and Dr. Howard Berg for the KAF95 strain, and Dr. Steve Block, Dr. Keir Neuman, and Dr. Rob Phillips for helpful discussions. Peter Lee was instrumental in designing the detector circuit. Dr. Peter Tarsa, Ricardo Brau, Jorge Ferrer, Carlos Castro, Ahmed Khalil, and Valeria Garbin assisted with construction, testing, and text revisions. The Biological Engineering 481 and 309 classes greatly helped to refine the instruments and modules. a Electronic mail: mjlang@mit.edu 1 A. Ashkin, “Acceleration and trapping of particles by radiation pressure,” Phys. Rev. Lett. 244, 156–159 1970. 2 M. J. Lang and S. M. Block, “Resource Letter: LBOT-1: Laser-based optical tweezers,” Am. J. Phys. 713, 201–215 2003. 3 M. J. Lang, P. M. Fordyce, A. M. Engh, K. C. Neuman, and S. M. Block, “Simultaneous, coincident optical trapping and single-molecule fluorescence,” Nat. Methods 12, 133–139 2004. 4 K. Visscher, M. J. Schnitzer, and S. M. Block, “Single kinesin molecules studied with a molecular force clamp,” Nature London 400, 184–189 1999. 5 A. Ishijima, H. Kojima, T. Funatsu, M. Tokunaga, H. Higuchi, H. Tanaka, and T. Yanagida, “Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin,” Cell 922, 161–171 1998. 6 C. G. Galbraith and M. P. Sheetz, “Keratocytes pull with similar forces on their dorsal and ventral surfaces,” J. Cell Biol. 1476, 1313–1324 1999. 7 S. P. Smith, S. R. Bhalotra, A. L. Brody, B. L. Brown, E. K. Boyda, and M. Prentiss, “Inexpensive optical tweezers for undergraduate laboratories,” Am. J. Phys. 671, 26–35 1999. 8 R. Pastel, A. Struthers, R. Ringle, J. Rogers, C. Rohde, and P. Geiser, “Laser trapping of microscopic particles for undergraduate experiments,” Am. J. Phys. 6811, 993–1001 2000. 9 D. N. Moothoo, J. Arlt, R. S. Conroy, F. Akerboom, A. Voit, and K. Dholakia, “Beth’s experiment using optical tweezers,” Am. J. Phys. 693, 271–276 2001. 10 J. Bechhoefer and S. Wilson, “Faster, cheaper, safer optical tweezers for the undergraduate laboratory,” Am. J. Phys. 704, 393–400 2002. 11 K. C. Neuman, E. H. Chadd, F. G. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to Escherichia coli in optical traps,” Biophys. J. 775, 2856–2863 1999. 12 http://www.openwetware.org/wiki/Optical_Trap 13 K. Svoboda and S. M. Block, “Biological applications of optical forces,” Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 1994. 14 M. P. Sheetz, “Laser tweezers in cell biology,” Methods Cell Biol. 55, 111–112 1998. 15 M. J. Lang, C. L. Asbury, J. W. Shaevitz, and S. M. Block, “An auto- 13 Am. J. Phys., Vol. 75, No. 1, January 2007 Appleyard et al. 13
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