Project Overview

Inspired by the need for a simple, inexpensive, and portable means of toxin detection, Brown University’s Team Toxipop wanted to create a biosensor that utilizes a novel method to detect the presence of a toxin in a solution of bacteria. After a great deal of brainstorming and research, our team finalized on a design for our project that interfaces both biological and electrical systems. Our design is primarily based on a list that our team compiled of ideal bio-sensor guidelines:

Biological

• Uses minimal biological machinery
• Direct induction of system by inducer creates a sensitive system
• Versatile construct

Engineering

• Compact and user-friendly system for sample analysis
• Economically feasible

Minimal biological machinery

Our biological construct consists of three interacting proteins under the control of a single promoter. Such a simple construct increases efficiency and minimizes sources of error in the system.

Direct induction of system by inducer optimizes sensitivity

The triggering of the lysis gene cassette is directly influenced by inducer presence, creating a sensitive system that should maintain a constant gain.

Versatile construct

The simplicity of our construct lends to its versatility. The promoter controlling the lysis cassette can easily be interchanged, optimizing our system's potential to detect several different types of toxins and other substances. For example, a mercury-induced, arsenic-induced, or lead-induced promoter, all of which have been introduced by iGEM teams in the past, could be placed in control of the lysis cassette, creating three separate and specific toxin detection systems.

Compact and user-friendly

The physical bio-sensor envisioned by our team would be hand-held, consisting of a small compartment of bacterial cells with electrical probes fixed in this compartment. The probes feed information to a simple circuit within the sensor which monitors changes in the current of the bacterial solution and outputs that change via a simple LED light signal (See the "Application" below for more details). This novel electrical reporting system would enable any person with little or no knowledge of the machinery of the bio-sensor to utilize it effectively. Additionally, in comparison to a physical detection system, in which the number and surface area of the probes are fixed and limited, with a biological system, there are billions of bacteria, each with the ability to sense.

Economically feasible

E. coli bacteria, which provide the main machinery of the bio-sensor, are inexpensive to obtain, grow, and store. Additionally, after consultation with mechanical engineers, we discovered that the circuit required for our proposed system would be simple to create and inexpensive to produce, in the range of a few dollars.

Application

The proposed biosensor

Current Water Contamination Machinery

The proposed prototype for Team Toxipop's biosensor is pictured on above on the left. It would be inexpensive to produce, portable, and user-friendly compared to a current water detection system, as pictured above on the right.

• The Toxipop biosensor consists of a small box with a compartment containing lyophilized E.coli bacterial cells, engineered with the lysis cassette under the control of a specific toxin-inducible promoter. Two graphite electrical probes are fixed on either side of this compartment (white arrows) which detect relative changes in current and feed that information to a small circuit board within the sensor. This circuit board, receiving a constant voltage from a battery source, will compute relative changes in resistance (or conductance) and will report that information via an LED light signal.

• The following protocol could be proposed for the Toxipop system:

1. Re-constitute lyohphilized cells with cell growth media.
2. Apply a few drops of the water sample with potential toxin into specified compartment.
3. Allow cells to to sit for approximately two hours. The white light will remain on during this time. Once the amount of time required for lysis has passed, either the red light will switch on, indicating the presence of the toxin, or the green light will switch on, indicating that no or very little of the toxin is present in the given water sample.

• This physical biosensor is a possibility for the future with the novel electrical reporting system designed by Team Toxipop this summer.

Other Applications

After some brainstorming, the team came up with some other possible applications for our electrical reporting system and our auto-lysis cassette:

• The electrical reporting system could provide a new and accurate means of measuring degree of protein expression. The relative change in current would directly correlate to the amount of protein expression. Unlike GFP, for example, which is a more boolean reporter of protein expression, our novel reporting system would be a quantitative and thus more specific method of reporting.

The auto-lysis device could have several applications, some of which are listed below:

• Use as a "kill-switch"
  • Keep all or certain cells engineered with the lysis cassette under an inducible promoter (ex. pBAD) so that if protein expression begins going out of control in a system, some arabinose can be added to the cells and they will kill themselves.

• Use as an antibiotic
  • Bacterial cells can be engineered to produce constitutively lysozyme and also engineered with the lysis cassette under an inducible promoter. When the cells are induced to lyse, they will release lysozyme into the surrounding solution, killing other bacteria.

• Possible use in biofilms