The overall goal of this experiment is to demonstrate a simple modified litmus test for bacterial detection. This method describes how an E.coli-activated RNA-cleaving DNA enzyme can be coupled with a urease-based signal trandsduction platform to generate a color change in response to E.coli. The two main advantages of this technique are the simplicity of the test and its applicability to other analyte-responsive RNA-cleaving DNA enzymes.
Demonstrating the procedure will be Seperh Manchehry and Dingran Chang, graduate students from Dr.Yingfu Li's lab at McMaster University. After preparing reagents and buffers according to the text protocol, prepare a stock solution of succinimidyl 4-cyclohexane-1-carboxylate, or SMCC, by dissolving one milligram of SMCC in 676 microliters of DMSO. Vortex and place on ice until use.
Dissolve one milligram of urease in milliliter of 1x PBS and also place that on ice until use. To synthesize urease DNA, or UrDNA, add 10 microliters of 100 micromolar LD1 to a 2.5 milliliter microfuge tube. Then, add 140 microliters of doubly-distilled water and 40 microliters of 10x PBS and vortex.
Next, add 80.5 microliters of SMCC stock and 159.5 microliters of DMSO. Then, vortex again and use a benchtop centrifuge to briefly spin. Incubate the reaction at 37 degrees Celsius in an incubator or a heat block for 60 minutes.
Following the incubation, add 200 microliters of 1x PBS, 60 microliters of three-molar sodium acetate, and 1.5 milliliters of cold 100%ethanol to the tube. Mix by vortexing, and incubate the reaction at minus 20 degrees Celsius for 30 minutes. Centrifuge the solution at 20, 000 times g and four degrees Celsius for 20 minutes.
Then, remove the supernatant and dry the pellet under vacuum. To the dried pellet, add 400 microliters of urease stock and incubate at room temperature for 5 hours. Transfer 200 microliters of crude conjugate to a pre-washed 100, 000 molecular weight cutoff centrifugal filter column.
Centrifuge the column at 14, 000 times g for five minutes. Transfer the remaining 200 microliters of crude conjugate to the column and centrifuge at 14, 000 times g for five minutes. Then, remove the column and place it upside down in a new two-milliliter microfuge collection tube.
Centrifuge the inverted column and tube at 1, 000 times g for two minutes. After removing the collection tube, add 30 microliters of 1x PBS to the centrifugal column to wash the membrane for additional recovery of conjugates. Invert the column again and place it back into the collection tube.
Centrifuge the column at 1, 000 times g for two minutes. Remove and dispose of the column. Store the UrDNA at four degrees Celsius until use.
To assemble an E.coli-activated RNA-cleaving DNAzyme, or EC1, and UrDNA onto magnetic beads, mix the magnetic bead stock well and transfer 100 microliters of bead suspension to a 1.5 milliliter microfuge tube. Then, place the tube on a magnetic rack holder for isolating the beads. Remove the supernatant by pipetting and add 150 microliters of binding buffer, or BB, to the tube.
Remove the tube from the holder and carefully tap the tube to re-suspend the beads into a homogeneous solution. After repeating the BB wash two more times with the beads in suspension with BB, add 10 microliters of 10 micromolar EC1. Carefully mix by tapping on the tube.
Incubate the solution with mild shaking for 30 minutes. To avoid aggregation of the beads, tap on the tube every two to three minutes. Next, place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.
Then, use 150 microliters of BB to wash the beads three times. Once the washing is complete, suspend the beads in a total of 150 microliters of BB.To this solution, add 15 microliters of UrDNA and heat the sample at 45 degrees Celsius for two minutes, then cool the solution to room temperature and incubate for two hours. Place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.
Add 100 microliters of reaction buffer, or RB, then remove the tube from the magnetic rack and carefully re-suspend the beads. After preparing E.coli cells according to the text protocol, pre-wash a 1.5 milliliter microfuge tube by adding and vortexing 100 microliters of RB.Then, discard the buffer. Add 15 microliters of assembled EC1 to the washed tube, then place the tube on the magnetic rack and aspirate the supernatant.
Remove the tube from the rack, add 100 microliters of RB, and carefully re-suspend the magnetic beads. After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E.coli sample to the tube and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.
After the incubation, add 90 microliters of doubly-distilled water and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5 milliliter microfuge tube. It is very important to pipette the solution carefully without taking up any of the magnetic beads.
These beads are loaded with urease and will generate a false positive signal. Add 15 microliters of 0.04%Phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.
The starting color of the litmus test must be yellow, reflecting a solution pH below 5.5. If necessary, the starting pH of the litmus test can be adjusted with a weak buffer, such as one millimolar sodium acetate buffer at pH five. This figure shows the results of the bacterial litmus test, where E.coli-activated RNA-cleaving DNAzyme EC1 was used as the DNAzyme and Phenol red was used as the pH indicator.
As shown by the lack of color change in the absence of E.coli, EC1 is highly specific and exhibits minimal activity towards other bacteria. The depth of color change is dependent both on the number of E.coli cells used in the DNAzyme activation step and on the time allowed for the urea hydrolysis step. More E.coli cells result in a stronger color change, and a longer time for urea hydrolysis allows for the detection of a smaller number of cells.
This graph shows the pH change as monitored using a handheld pH meter. The presence of 10 to the seven E.coli cells resulted in a gradual increase in pH by three units within 10 minutes. In contrast, the absence of E.coli did not cause detectable pH changes in the same conditions.
Once mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it is important to remember that urease is very active, and poor washing or contamination will lead to a false positive. Following this procedure, this method can be performed to test for E.coli in more relevant conditions, such as testing for environmental water quality.
After watching this video, you should have a good understanding for taking advantage of a simple litmus test for E.coli testing. Don't forget that this test can be used to detect pathogenic E.coli, which is extremely hazardous. You need to take biosafety training before conducting any of the tests for pathogenic bacteria.
