Project

Description

Our project is aimed at developing a method for degradation and detection of carcinogenic substances called polycyclic aromatic hydrocarbons, often abbreviated as PAHs. These are organic compunds formed as biproducts resulting from industrial practices such as: burning of fuel, wood, waste, polypropylene, polystyrene and petroleum products. PAHs are also dominant in petrol leakages and petrol industries. Hence, degradation of these compunds could contribute to solving one of the world's many environmental problems. Apart from environmental issues, PAHs are toxic, teratogenic and carcinogenic, as research proves that they cause skin, lung, bladder, liver, and stomach cancers in lab mice. Therefore, the importance of degrading these compunds is great.

Our priorities are to detect these substances in an automated manner. Four previous iGEM teams were interested in this topic and succeeded to some degree in degrading low-molecular weight PAHs. We plan to benefit from this and from our own team’s knowledge and target high-molecular weight PAHs as well.

Goal

First, we are working on developing a bacterium that will degrade high and low molecular weight PAHs. We aim to engineer biobricks that contain the necessary genetic information and then transfer it into Escherichia coli, a non-pathogenic bacterium. We are working not only to degrade PAHs, but also to provide an automated biosensor that can be used to detect the presence of these substances in different samples. The PAH biosensor can be used in food factories, waste management facilities, and water treatment plants. With our detector we can encourage industries to monitor their PAH emissions.

The construct

In our construct we strive to degrade both heavy molecular weight PAHs, such as Benzo(a)pyrene, as well as low molecular PAHs, such as naphthalene. The low molecular weight PAHs can freely diffuse over the bacterial cell membranes, while the heavy molecular weight PAHs diffuse at a lesser degree. PAHs found as pollutants are always found in heterogeneous mixes of heavy and low molecular weight compounds. In our system, one of the light weight PAHs, naphthalene, will be degraded by six enzymes acquired from a naphthalene-degrading plasmid from Pseudomonas putida. These enzymes will degrade naphthalene into the smaller and less toxic compund salicylate, which in turn will bind to the inducer NahR, acquired from the same pathway. The NahR inducer has been utilized to activate the production of two enzymes, a laccase and a dioxygenase. These two enzymes will be exported out of our cell in order to degrade the heavy-weight BaP. Since BaP will not freely enter the cell we have attached export tags so our enzymes could work extracellularly. NahR will also induce the production of a fluorescent protein to give a visual indication of the presence of PAHs in the medium.

To further optimize our construct, we have also included the expression of biosurfactant-producing enzymes RhlA and RhlB. As the PAHs are highly hydrophobic they tend to aggregate in an aqueous solution, making it difficult for the active sites of the degrading enzymes to reach their substrates. The biosurfactants will dissolve these aggregates, freeing up more PAHs for the enzymes to degrade.