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.

The design of the project stayed largely the same, but we modified and altered the specifics and implementation as we continued with our cloning and lab work, specifically our experiments testing HRP.

Our initial plan was to use hydrogen peroxide as our chromogenic substrate, as when in the presence of HRP a red color change is observed. Our initial tests using HRP and hydrogen peroxide did not result in visible color change with the amount of HRP we anticipated being present at the lower detection limits of our test.

Subsequently, we turned to testing more sensitive substrates. In particular, we performed test using the chemiluminescent substrate Luminol and hydrogen peroxide with HRP to confirm that this substrate combination would be usable for applications of our testing system that required more sensitive detection (that data can be found under our Results page).

As we required a chromogenic substrate that would produce a visible color change for use of our test kit by the general public, we went searching for the perfect substrate. With the help of Dr. Rob Balza, we landed upon using tetramethylbenzidine (TMB) which Dr. Balza was kind enough to provide for us.

We then performed tests using TMB and were able to observe visible color changes that would be readable by the average consumer! The color change indicating HRP cleavage of the substrate could also be observed within 5 minutes indicating our test kit would be faster than any current E.coli test available on the market. The color change data can be found on our Results page.

Our system design further changed as we were attempting to design, or locate, an adapter that would open to expose a piece of 0.22um filter paper and could be attached via a luer lock to a syringe. Our adviser Dr. Werner found a simple system that fit our requirements but unfortunately did not arrive in time to perform tests confirming its suitability to our application.

As a result, we decided to perform tests using a vacuum apparatus as a substitute for the syringe system. The same type of filter paper was used and tests that mimicked the conditions we expected our kit to be used under were performed. The data from those can be found on our Results page.

In the end, we re-worked our initial system protocol and design 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 for approximately 5 minutes.
• After incubation, the solution was 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.
• 5% TMB substrate is then applied to the filter paper and allowed to sit for 5 minutes. The HRP bound to the J-protein and by extension any E. coli from the water sample, cleaves the TMB resulting in a green to yellow color.
• The filter paper is removed from the adapter, and the color of the signal can be compared to a prepared color scale that indicates the relative concentration of E. coli in the original water sample.