In order to determine the bending rigidity of individually coli cells, first, grow the bacteria to exponential phase and then attach them to a chemically treated cover slip surface. Let the cells already attach to the surface grow further so that they develop flexible ends. Next flow in dielectric beads, trap a bead and bend it to the flexible tip of a cell.
The final step of the procedure is to bend the cell with the optical trap and measure the resulting forced displacement data. The bending stiffness of the cell can be calculated by a linear fit to the forced displacement data. Hi, I'm Steven Wong from the Lab of Professor Joshua Shere in the Louis Sickler Institute of Integrative Genomics and the Department of Physics at Princeton University.
Today we'll show you in a procedure of bending individual eco cells and the marrying cell stiffness. We use this procedure in our lab to study the contributions of different intracellular structures to the mechanical integrity of the bacteria cells. So let's get started.
To begin this procedure, grow three milliliters of any e coli cells in LB medium to exponential phase. Once the cells reach an optical density at 600 nanometers of between 0.2 and 0.4, supplement the culture with 50 micrograms per milliliter of cephalexin for 15 minutes to induce filamentous growth. During these 15 minutes, make a flow chamber.
Attach two pieces of double-sided tape with a three millimeter gap between the pieces to a glass slide and tape a cover slip on top. The gap between the pieces of tape creates a channel for flowing liquid. Coat the cover slip with PEI by flowing 10 microliters of 1%polyethaline diluted in water into the flow chamber with a micro pipette incubate for five minutes.
Then wash by flowing in 200 microliters of water at one end of the chamber and collecting at the other end with a vacuum. After 15 minutes of growing the bacteria in the presence of cephalexin, spin down one milliliter of cells in a micro centrifuge and resuspend in 200 microliters of LV with cephalexin. Next, again, use a micro pipette on one end and vacuum on the other end to flow 50 microliters of the concentrated cell culture into the chamber holding the coated cover slip incubate for three minutes.
Then wash with 50 microliters of Vel B supplemented with cephalexin. To remove unattached cells, incubate the chamber at 37 degrees Celsius for 30 minutes to one hour to allow the attached cells to grow before placing them on the optical trapping instrument while the cells are incubating. Prepare polylysine coated beads by incubating three microliters of 0.5 micrometer diameter polystyrene beads in one of 0.1%Polylysine diluted in water for 30 minutes.
When the beads finish incubating, wash them three times by spinning down in a micro centrifuge and resus suspending in one milliliter of water, proceed to bind the beads to the bacteria and measure the binding stiffness of the cells. The most difficult aspect of this protocol is being able to rec, locate, and recognize a good cell for the measurements of bending, stiffness. And a good cell is one with a well-defined stuck end and free end Place.
The flow chamber on the microscope look for a cell with a well-defined stuck end. Some cells are attached just at one tip, and the bending force at the other tip leads to a whole cell pivoting rather than bending. To find a suitable cell, bend each cell quickly with the trap by using the joystick controlled stage motion.
When a suitable cell is found, dilute the bead solution by a factor of two into lb with cephalexin, add 30 microliters of the bead solution to the flow chamber by pipetting into one end and gently flowing out at the other end with a Kim wipe tissue optically trap a floating bead and touch it to the free tip of a cell. Wash the chamber again by micropipet and using a Kim white tissue with 50 microliters of cephalexin in LB to remove unattached beads. Next, run a custom written lab view program to apply bending forces to the cell and record the forced displacement data.
First, calibrate the detector response by RA scanning the attached bead within the detection laser beam and recording the 3D PSD voltage signals. Then identify the long axis of the cell in a microscope image. After identifying the long cell axis, move the cell in steps in a direction perpendicular to the long axis while moving the cell record, the distance moved and the displacement of the bead from the center of the optical trap.
Finally, convert the displacement values to applied force using the previously measured trap stiffness and save. The tip displacement and force to file here are forced displacement data for a single cell. The slope of this line is the bending stiffness of the cell.
We've just shown you how to marry the bending stiffness of e coli with an optical trap. When using this procedure, it's important to remember to find a good cell with a well-defined stuck end and free end. So that's it.
Thanks for watching and good luck with your experiments.
