Team:Edinburgh

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iGEM PROJECT "TNT/RDX Biosensor and Bioremediator"

In 2007, 5 426 new casualties were recorded from landmine explosions. 71% of these casualties were civilians. A further 46% of the civilian casualties were children^[1]. This made us realise the need for the production of a cheap, safe and accurate method that can be applied in a big scale to help detect landmines. A synthetic organism could be just what is needed.

Even though landmines are buried under soil, they normally leak indicating their imminent position with a chemical fingerprint. TNT-filled landmines produce three major source chemicals, namely 1,3-DNB, 2,4-DNT, and 2,4,6-TNT ^[2]. In addition, the natural degradation of explosive compounds, such as TNT, by bacterial enzymes produces nitrogen in the form of Nitrites^[3]. Nitrites are also one of the by-products of the degradation of another explosive used in landmines, namely RDX. In the latter case, this can be achieved by the soil bacterium Rhodococcus rhodochrous^[4].

Our project is concerned in making a biosensor that would detect both the presence of TNT and nitrites/nitrates.

Natural nitrite concentration in soil tends to be very low (below 0.1 mg NO2-N /kg)^[7]. Thereby the possibility of false positive results decreases. Our biosensor would also detect nitrates but these would need to be at a much higher concentration than nitrites to bring about a response.

The fact that excessive fertilisation with ammonium producing fertilizers such as urea can cause an increase in the presence of nitrites in the soil ^[5][6][7] gives the possibility that our device can be used in diverse fields of interest, from landmine identification (ranging from TNT landmines to RDX ones), to assaying extent of fertiliser induced nitrite/nitrate pollution.

Please read our detailed project description and part characterisation for further details.

References [1] Landmine monitor 2009. Landmine Monitor Fact Sheet. Landmines and children. http://www.lm.icbl.org/index.php/content/view/full/24160

[2] Thomas F. Jenkins, Daniel C. Leggett, Paul H. Miyares, Marianne E. Walsh, Thomas A. Ranney, James H. Cragin and Vivian George. 2001. Chemical signatures of TNT-filled land mines. Talanta. 54 (3):501-513

[3] Christopher E. French, Stephen Nicklin, and Neil C. Bruce. 1998 Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase. Applied and Environmental Microbiology, 64 (8): 2864-2868

[4] Seth-Smith, H.M.B., Rosser, S.J., Basran, A., Travis, E.R., Dabbs, E.R., Nicklin, S. andBruce, N.C. 2002. Cloning, Sequencing and Characterization of the Hexahydro-1,3,5-trinitro-1,3,5-triazine Degradation Gene Cluster from Rhodococcus rhodochrous. Appl. Environ. Microbiol. 68:4764-4771.

[5] Smith, C.J. & Chalk, P.M. 1980. Gaseous nitrogen evolution during nitrification of ammonia fertilizer and nitrite transformation in soils. Soil Science Society of America Journal, 44, 277–282.

[6] Burns, L.C., Stevens, R.J. & Laughlin, R.J. 1995. Determination of the simultaneous production and consumption of soil nitrite using 15N. Soil Biology and Biochemistry, 27, 839–844.

[7] Van Cleemput, O. & Samater, A.H. 1996. Nitrite in soils: accumulation and role in the formation of gaseous N compounds. Fertilizer Research, 45, 81–89.

Why we differ?

Existing systems

Biological systems for mine and particularly TNT detection have been described previously, the most prominent of which is the patented Microbial Mine Detection System (MMDS) developed by Dr Paul Burlage and the Oak Ridge National Laboratory. The MMDS is normally a bacterium able to detect and exhibit chemotaxis towards TNT and the vapours released as a result of its degradation, in particular nitrates and nitrites. The bacteria utilized at the Oak Ridge Laboratory are mainly the Bacillus or Pseudomonas species, such as Pseudomonas putida, which have been found to naturally exhibit growth towards a source of TNT degradation. Although the MMDS patent states that the recombinant microorganism is able to directly detect TNT, it is likely that the bacteria do not exhibit chemotaxis towards TNT per se but rather the organism is sensitive to high levels of nitrate and nitrite. The sensitive genes are in turn fused with a Green Fluorescent Protein (GFP) reporter gene, making it possible to visualize the fluorescent bacteria with the help of a UV illuminator. The MMDS has been field tested at a South Carolina site, where the system was able to locate all five of the hidden mine sites, showing the great potential of biological mine detection (Fig. 1).

Figure 1. An image showing a UV scan of a 3x4 metre field test area, demonstrating higher fluorescence levels around the mine site as well as plant material, possibly due to TNT being absorbed through the root system of surrounding plants. Image source

Advantages of our system

The primary advantage of our system over the MMDS is the specificity of the TNT detection system. Beside the nitrate and nitrite sensitivity, the MineBusters system (MBS) is able to exhibit high specificity to TNT with the help of a completely novel computationally designed TNT-specific promoter. Prior to this creation TNT specificity has not been found to occur in nature. Due to this the MBS is able to provide much more accurate readings of mine presence. With the help of the enzyme nitroreductase the MBS is also able to degrade TNT, providing further signal for the system. Most importantly, the MBS signals the presence of TNT as a combination of light and fluorescence-emitting systems, providing a directly visible outcome of various colours and in this way eliminating the need for any scanning or illumination equipment. We believe that on the basis of this our system has the potential to be much more accurate, easier and cheaper to operate than the existing one. We are confident that our system is a great candidate for a cheap, accessible and easy to use mine detection and eradication method^[references]