Overview
This video demonstrates a colorimetric E. coli detection method, involving an E. coli biomarker activating a DNAzyme, leading to cleavage of RNA linkage and detachment of a DNAzyme-urease conjugate. A subsequent colorimetric assay detects the E. coli biomarker through ammonia-induced pH changes.
Protocol
1. Preparing E. coli Cells for Testing
- For a desired cell suspension, transfer 1 ml of cultured stock to a 1.5 ml microfuge tube.
- Centrifuge the cells at 6,000 x g for 10 min at 4 °C. Carefully remove the supernatant without disturbing the cell pellet.
- Add 10 µl of reaction buffer to the cell pellet and resuspend the cells. Sonicate the cell suspension for 5 min. Transfer the cell suspension to an ice box for 5 min.
- Sonicate the cell suspension for another 5 min.
- Centrifuge the cell suspension at 13,000 x g for 10 min at 4 °C. Use the supernatant for testing (10 µl).
2. Litmus Test
- In a 1.5 ml microfuge tube, prewash the tube by adding and vortexing 100 µl of reaction buffer (RB) in the microfuge tube and discarding the buffer.
- Transfer 15 µl of assembled EC1 to the washed microfuge tube.
- Wash the magnetic beads by placing the microfuge tube on a magnetic rack. Remove the supernatant by pipetting. Remove the microfuge tube from the rack, add 100 µl of RB, and carefully resuspend the magnetic beads.
- Wash the MB two more times by repeating step 2.3.
- Place the microfuge tube back on the magnetic rack, remove the supernatant, and add the 10 µl E. coli sample prepared from step 1.3.
- Mix the sample and magnetic beads carefully by gently tapping on the microfuge tube.
- Incubate the reaction at room temperature for 1 hr.
- To the reaction, add 90 µl of ddH2O and place the microfuge tube onto a magnetic rack.
- After approximately 3 min of magnetic separation, carefully transfer 85 µl of the supernatant to a 0.5 ml microfuge tube. Withdraw the supernatant slowly to avoid collecting any magnetic beads.
- To the above microfuge tube add 15 µl of 0.04% phenol red and 100 µl of substrate solution.
- Take a photograph at specific time intervals to record color change.
NOTE: The change in pH can also be monitored using a pH meter with a microelectrode. The starting pH should be approximately 5.2-5.5 (the solution is yellow). If not, the solution can be adjusted by the addition of 1 mM Acetate Buffer pH 5.0).
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Materials
Name | Company | Catalog Number | Comments |
Ethylenediaminetetraacetic acid (EDTA) | VWR AMRESCO | 105 | |
Sodium Hydroxide (NaOH) pellets | BIO BASIC CANADA INC | SB6789 | |
Tris-base | VWR AMRESCO | 497 | |
Urea | VWR AMRESCO | M123 | |
Xylenecyanol FF | SIGMA-ALDRICH | X-4126 | |
0.04% Phenol red | SIGMA-ALDRICH | P3532 | |
10x T4 polynucleotide kinase reaction buffer | Lucigen | 30061-1 | |
E. coli K12 (MG1655) | ATCC | ATCC700926 | |
Magnetic Bead (BioMag) | Bangs Laboratories Inc | ||
Magnetic Seperation Rack | New England BioLabs | S1506S | |
Microfuge tubes | Sarstedt | 72.69 | |
Cell culture incubator | Eppendorf Scientific | M13520000 | |
Branson Ultrasonic cleaner | Branson | N/A |