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From 2013hs.igem.org

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Revision as of 05:13, 1 April 2013

Preliminary Introduction/Abstract

Introduction

In rural homes with no connection to a main sewage line, a septic tank is a common solution for sewage treatment. In North America, about 25% of the population relies on a septic tank. However, the accumulation of waste products can lead to high levels of nitrates, phosphates, and sulfates in the septic tank. Because the tank represents an anaerobic environment, fermentation takes place. As a result, the redox potential of the septic tank is quite low, meaning that sulfates are reduced to hydrogen sulfide, and nitrates and phosphates, even in high concentrations, remain highly solubilized. The production of these chemicals can have a number of negative ramifications: hydrogen sulfide, for example, is toxic to humans; and high concentrations of nitrates and phosphates in soil or water cause eutrophication. Recent studies have also found that high nitrate and phosphate concentrations in water can cause damaging effects to the marine wildlife in the ecosystem. There is currently no method for analyzing the contents of a septic tank, other than pumping it out. If such a process were developed, it could potentially prevent costly septic tank repairs and environmental hazards that result from an improperly functioning septic tank.

Solution Statement

The goal of this study, therefore, is to develop a multi-input logic gate in Escherichia Coli, which can detect the presence of a number of these environmentally degrading compounds, and for each, produce a unique colorimetric output. We hope to apply the principles of electrical engineering by using the Google ADK to sense this colorimetric output and send a notification through email. We envision that for an end-user, our elegant synthetic biology solution will allow a homeowner to easily and effectively be notified of the need for inspection of their septic system.

Methods

Our multi-input logic gate will produce a colorimetric output for nitrates, phosphates, and toxic chemicals such as hydrogen sulfide and sodium hypochlorite, which are all detrimental to the function of septic tanks. The diagram below shows the genome of our E. Coli plasmid that will produce a unique colorimetric output based on the presence of the targeted metals in the local environment.

Figure 1. MG1655 E. coli plasmid after insertion of the genetic elements necessary for expression of fluorescent proteins based on presence of phosphates, nitrates, or toxic chemicals. Two of the three FPs in the plasmid are regulated by a different promoter that is activated in the presence of the targeted substance. Activation of the CreA (phosphate activated) and PyeaR (nitrate activated) promoters results in the translation of GFP and BFP proteins, respectively. The third FP, mCherry (red FP), is transcribed constitutively, such that any chemical toxic enough to kill the E. coli (such as hydrogen sulfide or sodium hypochlorite) will result in a detectable decrease in this color. The appropriate FP genes will be inserted at the BamI restriction site, as shown. The Ampicillin Resistance Gene allows positive selection of plasmids that have been transformed as intended, and is inserted at the EcoRI site as shown.

To sense these colorimetric outputs, the Google ADK (Accessories Development Kit) can be used. The Google ADK can be modified to detect the colorimetric outputs from the E. coli plasmid. The device monitors color by detecting the amount and type of light absorbed across a known difference. A prototype of the device is shown in Figure 2. The chemicals present and their approximate concentrations based on absorbance of color are then sent through the Google ADK to the septic tank owner to notify them that an inspection of the tank is needed. In addition, if the red color that is continually produced by the E. coli bacteria is diminished, the modified Google ADK will notify the septic tank owner to recommend tank inspection, as this decrease in mCherry protein is a result of chemicals (ex. sodium hypochlorite, a key ingredient in bleach) killing the E. coli. The Google ADK will serve as a detector of the colorimetric output and serves the main purpose of communicating data to the tank owner.

Figure 2. A proposed design for the system is shown above. The Google ADK device allows for the detection of minute changes in color concentration, which result from changes in the expression of different FPs from the E. coli plasmids. This information can be sent to the owner, in the form of a recommendation for inspection of the tank. The entire system is surrounded by waterproof plexiglass to protect the device from any substances in the septic tank.