• Enzymatic degradation
• Naphthalene
• Biosurfers
• Software
• Biosensor design

Project

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Overview and aim

This is a project in synthetic biology performed during a summer by 24 dedicated and curious students from Uppsala University who planned the project over the entire spring semester – and in the fall the team will present their achievements in Boston where iGEM teams from all over the world compete with their projects. We have decided to develop a method for degradation and detection of the carcinogenic substances polycyclic aromatic hydrocarbons (PAH:s), thus contributing to solve one of the world's many environmental problems. Our priorities are to detect those substances in an automated manner and to optimize enzyme efficiency, which will result in one distinct product. Four previous iGEM teams were interested in this topic and succeeded to some degree in degrading low-molecular weight PAH:s. We plan to benefit from this and from our own team’s knowledge and target high-molecular weight PAH:s as well.

Vision

PAH:s can result from industrial practices such as: burning of fuel, wood, waste, polypropylene, polystyrene and petroleum products. Industries that use coal tar, coke or asphalt are also a target for our products. Moreover, PAH:s are dominant in petrol leakages and petrol industries. Since there is always a search for a better alternative or a more effective way to degrade the PAH:s, our product will be a pioneer in the market.

We aim to provide a microorganism that can degrade high- and low-molecular weight PAH:s. These substances are formed during incomplete combustion or pyrolysis of organic materials and can be found in water, soil, and food products. According to researches, PAH:s cause skin, lung, bladder, liver, and stomach cancers in lab mice. PAH:s can also affect the hematopoietic and immune systems generating reproductive, neurologic and developmental effects. As a result, degrading these substances is an urgent necessity.

The construct

In our construct we strive to degrade both the heavy molecular weight PAH:s, such as benzo(a)pyrene (BaP), as well as low molecular PAH:s, such as naphthalene. The low molecular weight PAH:s can freely diffuse over the bacterial cell membranes, while the heavy molecular weight PAH:s diffuse at a lesser degree. PAH:s found as pollutants are always found in heterogeneous mixes of heavy and low molecular weight. In our system the light weight PAH, naphthalene, will be degraded by six enzymes acquired from a naphthalene-degrading plasmid from Pseudomonas putida. These enzymes will degrade the naphthalene into the smaller and less toxic 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 to these two enzymes in order to effectivize our system. The NahR system will also induce the production of a reporter gene such as sYFP to give a visual indication of the presence of PAH:s in the medium.

To further optimize our construct, we have also included the expression of biosurfactant-producing enzymes RhlA and RhlB. As the PAH:s 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 PAH:s for the enzymes to degrade.

The goal

First, we are working on developing a bacterium that will degrade PAH:s. We aim to engineer a biobrick that contains the necessary genetic information and then transferring it into E. coli, which is 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.