Team:WLC-Milwaukee/Applied Design
System Design
Introduction
The goal of our project is to produce a quick, easy, and cheap method of detecting E. coli in a water sample. This has a large number applications in industry and at home. To create a product, we are exploiting the mechanism that allows bacteriophages to bind specifically to E. coli. The portion of the J-protein from Lambda phage that is necessary for binding to E. coli was cloned and Histidine-tagged. This will allow us to purify the protein and conjugate the horseradish peroxidase enzyme to it. We can then expose water samples containing varying concentrations of E. coli to the HRP-conjugated J-protein (J-protein-HRP) and determine how much binds, thereby telling us how much E. coli is present, by measuring HRP activity.
Our initial system concept was based on the work performed by the 2017 WLC-Milwaukee iGEM Team, with the graphic used by permission from past team members. Additional brainstorming with Dr. Dan Ebeling came to the conclusion that with slight modification, the general design of the system would be maintained.
The kit concept and initial design is as follows:
- The sample of water to be tested is drawn up into a syringe preloaded with our lambda phage J-protein conjugated to Horseradish peroxidase
- The sample and J-protein-HRP conjugate solution is allowed to incubate.
- After incubation, the solution will be run through a 0.22um pore size filter to remove excess unbound J-protein-HRP conjugate to reduce false positive results.
- If E. coli was present in the water sample, the J-protein-HRP conjugate will have bound to the LamB outer membrane protein of E. coli and be caught with the bacteria on the surface of the filter paper.
- Chromogenic substrate (or chemiluminescent substrate depending on end user) is then applied to the filter paper. The HRP bound to the J-protein and by extension any E. coli from the water sample, then cleaves the chromogenic or chemiluminescent substrate resulting in a signal.
- Visible signal in the form of insoluble products of the chromogenic substrates can then be compared to a prepared color scale that indicates the relative concentration of E. coli in the original water sample.
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