Difference between revisions of "Team:Uppsala/Project"

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    <h2 class="header">Description</h2>
 
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     Our project has been aimed to develop a method for degradation and detection of the carcinogenic substances polycyclic aromatic hydrocarbons (PAHs), which results 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, breaking of these PAHs could  contribute to solve one of the world's many environmental problems. Apart from environmental issues, researchers prove that PAHs caused skin, lung, bladder, liver, and stomach cancers in lab mice. So, the importance of degrading these substances is in dire need.  
 
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    Our priorities are to detect these 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 PAHs. We plan to benefit from this and from our own team’s knowledge and target high-molecular weight PAHs as well.
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    <h2 class="header">Goal</h2>
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    First, we are working on developing a bacterium that will degrade high and low molecular weight PAHs. 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.
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    <h2 class="header">The construct</h2>
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    In our construct we strive to degrade both the heavy molecular weight PAHs, such as BaP(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. In our system the light weight PAHs, 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 so our enzymes could work extracellularly. This NahR will also induce the production of a fluorescent protein to give a visual indication of the presence of PAHs in the medium.
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    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.
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Revision as of 08:43, 27 July 2015

Project

Description

Our project has been aimed to develop a method for degradation and detection of the carcinogenic substances polycyclic aromatic hydrocarbons (PAHs), which results 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, breaking of these PAHs could contribute to solve one of the world's many environmental problems. Apart from environmental issues, researchers prove that PAHs caused skin, lung, bladder, liver, and stomach cancers in lab mice. So, the importance of degrading these substances is in dire need.

Our priorities are to detect these 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 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 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.

The construct

In our construct we strive to degrade both the heavy molecular weight PAHs, such as BaP(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. In our system the light weight PAHs, 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 so our enzymes could work extracellularly. This 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.