E Coli RP 4 37 are introduced into a chemotaxis device where they're exposed to defined spatial concentration. Gradients cells entering the chamber encounter the midpoint of the concentration gradient. Depending on whether the signal is perceived as an attractant or repellent bacteria rapidly move either up or down the concentration gradient.
Hi, I'm Derek Engler from the Laboratory of Molecular Systems Biotechnology and the Department of Chemical Engineering at Texas a and m University. Today we'll show you a procedure investigating bacterial chemo taxes in a microfluidic device. We use this procedure in our laboratories to study how e coli RP 4 37 migrates in concentration gradient of nickel sulfate.
So let's get started. The fluidic layer and the control layer are made by replica molding of Polymethyl SUBOXANE or PDMS from the SU eight master. To begin this procedure, mix the PDMS pre polymer and crosslinker at a 10 to one weight ratio.
Degas the mixture in a desiccate for one hour until air bubbles are removed. When the PDMS mixture is ready, place the SU eight master in a Petri dish and carefully pour the mixture on top of the SU eight master to the desired thickness Heat. The Petri dish containing the PDMS and the SU eight master mold at 80 degrees Celsius for two hours to cure the PDMS.
After two hours, remove the cured PDMS covered SSU eight master from the hot plate and peel the PDMS mold from the SU eight master. The desired structure will be embedded in the PDMS mold using a 20 gauge blunt end needle punch four holes in the PDMS mold for tubing to the gradient generator, cell inlet, and cell outlet. Now we are ready to assemble the PDMS device to bond the PDMS device.
First, clean a glass microscope slide with isopropanol and try it with an Airstream. Next, expose the PDMS mold and glass slide to oxygen plasma in a plasma etcher for about 30 seconds. Bring the PDMS mold into contact with the glass slide Heat the contacted slide and PDMS mold to 65 degrees Celsius on a hot plate for 15 minutes.
To assemble the PDMS device, use a razor blade to cut tubing at a 45 degree angle to the correct length required depending on the microscope setup. Then using forceps. Insert one end of the tubing into one of the four holes punched in the device.
Insert a blunt 30 gauge needle hub into the other end of the tubing. Repeat insertion of tubing and needle hub for all remaining holes. Load one milliliter of chemo taxes, buffer or CB in a three milliliter syringe.
Taking care to remove air from the syringe. Add CB to the outlet needle hub using a pipette and tap the needle hub to remove any air bubbles. Push a little CB out of the tip of the three milliliter syringe and connect the syringe to the needle hub Without trapping air.
Push CB through the device until all remaining needle hubs are filled with cb. This should remove a majority of the air bubbles. Next, fill two 500 microliter syringes with CB containing the appropriate concentrations of the chemo effector being tested.
Eliminate air bubble formation by pushing out a small drop of the syringe contents and touching the drops of the liquid in the needle hub and attaching the syringe to prepare highly motile bacteria. For the chemotaxis experiment, grow an overnight culture of e coli RP 4 37 with a GFP expression, plasmid in tripton broth or TB at 32 degrees Celsius with shaking the next day. Use the overnight culture to inoculate a 20 milliliter culture of TB in a 250 milliliter erlenmeyer flask to an optical density at 600 nanometers of about 0.05.
Grow the culture at 32 degrees Celsius with shaking when the cells are at an OD 600 of about 0.35 to 0.45. Harvest them by low speed centrifugation resus suspend cells to an OD 600 of about 0.35 in CB containing the chemo effector at the concentration expected in the middle of the channel. Finally, add RFP labeled dead cells at no D 600 of about 0.35 to the GFP expressing live cells.
These dead cells will be an internal control to ensure that any migration is not due to flow effects. The cell suspension is now ready to be loaded into the microfluidic device for the chemotaxis experiment. To begin the chemotaxis experiment, remove some of the CB from the cell inlet needle hub refill with the cell suspension.
Gently fill a 50 microliter syringe with the resuspended cells. Do this slowly as flagella can be sheared, which will reduce motility. Attach the 50 microliter syringe to the inlet needle hub as shown previously by pushing out a small drop of the syringe contents and touching the drop to the liquid in the needle hub and attaching the syringe position the device on the stage of a fluorescence microscope equipped with a high speed camera for image acquisition.
Place both the inlet syringes into the syringe pump and initiate flow such that the gradient is formed and cells are flowing through the tubing. Once cells enter the chemo taxes chamber wait about 20 minutes for the system to stabilize. Before imaging collect live green and dead red fluorescence images at different locations along the length of the chamber.
Typically we collect 100 images at each location at three second intervals. The fluorescence images acquired can be analyzed using any commercially available image analysis program such as Image J or metamorph, or using simple codes written in matlab. In this example, we'll use MATLAB codes.
Start by removing the background pixels in the image through this set threshold pixel intensity, and remove all pixels that have intensity less than the threshold. This minimizes noise. In the analysis, use the position of the dead cells to determine the center of the image.
This represents the position where cells entered the chemotaxis chamber and where they would be detected in the absence of any migration. Next, locate live cells in the image, divide the image into channels and determine the number of live cells in each channel. In our prior work, the 10 50 micrometer wide chemo taxes chamber was divided into 64 channels that were each about 16 micrometers wide.
After repeating these steps for all images, some of the total cell counts for each channel across all images. This gives the total cell count detected at each channel over the duration of the experiment. Calculate the chemo taxes, partition coefficient or CPC, which represents the direction of migration.
By performing the following steps, assign a multiplier of plus one and minus one to each cell located in the high concentration or right and low concentration or left sides of the dead cells respectively. Add up all the multiplied values and to normalize to the total number of detected cells. Calculate the chemo taxes, migration coefficient, or CMC, which weighs the migration of cells by the distance traveled.
A cell that moves to the farthest high concentration position channel 64 is given a weighting factor of plus one, whereas one that moves halfway into the higher concentration side is given a weight factor of plus 0.5 and a cell. Moving to the farthest low concentration position is weighted by minus one. Some of all the weighted cell counts and normalized to the number of cells to generate the CMC.
This graph shows a linear concentration gradient or profile formed in the microfluidic device using fluorescein isothiocyanate. The formation of a concentration gradient is demonstrated in this figure using blue and yellow color dyes. The cell inlet was CA so that there was no flow through it, and it can be seen that a range of colors between blue and yellow is formed.
Here are pseudocolor images from a representative chemotaxis experiment in which e coli RP 4 37 was exposed to a gradient of zero to 100 micromolar L aspartate or zero to 225 micromolar nickel sulfate in the device. The migration of live green bacteria towards al aspartate or away from nickel was imaged every 2.5 seconds for 30 minutes. Dead bacteria shown in red served as the control for flow effects in the device.
The spatial distribution of RP 4 37 in the microfluidic device in the absence of a gradient is shown in this graph in comparison to its response to an aspartate gradient and a nickel sulfate gradient. We've just shown you how to monitor the chemo taxes of e coli RP 4 37 in a gradient of nickel sulfate. When doing this procedure, it's important to remember to minimize vortexing of the cells so that a high motility subpopulation can be used.
So that's it. Thanks for watching and good luck with your experiments.
