Team:UC Davis/Project/Catalyst

From 2012.igem.org

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<h1> Cutinase </h1>
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<h1> Modules </h1>
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The iGEM team at UC Davis is working to produce a catalyst capable of degrading the common plastic, PET. Further description to come.
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We have created several modules and biobrick parts for the degradation and utilization of PET.
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<hp>Background</hp>
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<hp>LC-Cutinase and Initial PET Degradation</hp>
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We began the summer looking for a way to engineer <i>E. coli</i> to degrade plastics. We focused our attention on PET once we came across a paper that conducted a metagenomic analysis of leaf-branch compost, identified a cutinase homolog, and demonstrated its PET-degrading activity [1]. It was also found that the PET was broken down into two by-products: ethylene glycol and terepthalic acid (TPA).
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When looking for a catalyst capable of breaking down PET, we came across a paper that conducted a metagenomic analysis of leaf-branch compost, identified a cutinase homolog, and demonstrated its PET-degrading activity [1]. It was found that this catalyst broke PET down into two by-products: ethylene glycol and terepthalic acid (TPA).  
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<br>(pathway.jpg)
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We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and have placed it in the following construct.
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<br>(construct.jpg)
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<br>We decided to use pelB as a leading sequence on the cutinase gene since it has been shown to direct the enzyme to the periplasmic space [1] . Once the enzyme is led towards the membrane, there is a leakage that helps it be secreted into the extracellular matrix [1]. We hoped that this sequence would help the enzyme be secreted so the PET would more easily be degraded. When we ordered the cutinase sequence, we added pelB to the front of the sequence, in hopes of repeating the secretion shown in previous results.
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<hp>What we're doing</hp>
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<hp>Ethylene Glycol Modules</hp>
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We are looking to overproduce the LC-Cutinase gene in <i>E. Coli</i> so that we can further characterize its ability to degrade PET.
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<br>We decided to use pelB as a leading sequence on the cutinase gene since it has been shown to direct the enzyme to the periplasmic space [1] . Once the enzyme is led towards the membrane, there is a leakage that helps it be secreted into the extracellular matrix [1]. We hoped that this sequence would help the enzyme be secreted so the PET would more easily be degraded. When we ordered the cutinase sequence, we added pelB to the front of the sequence, in hopes of repeating the secretion shown in previous results.
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Revision as of 22:51, 28 August 2012

Team:UC Davis - 2012.igem.org


Modules

We have created several modules and biobrick parts for the degradation and utilization of PET.

LC-Cutinase and Initial PET Degradation
When looking for a catalyst capable of breaking down PET, we came across a paper that conducted a metagenomic analysis of leaf-branch compost, identified a cutinase homolog, and demonstrated its PET-degrading activity [1]. It was found that this catalyst broke PET down into two by-products: ethylene glycol and terepthalic acid (TPA).
(pathway.jpg)
We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and have placed it in the following construct.
(construct.jpg)
We decided to use pelB as a leading sequence on the cutinase gene since it has been shown to direct the enzyme to the periplasmic space [1] . Once the enzyme is led towards the membrane, there is a leakage that helps it be secreted into the extracellular matrix [1]. We hoped that this sequence would help the enzyme be secreted so the PET would more easily be degraded. When we ordered the cutinase sequence, we added pelB to the front of the sequence, in hopes of repeating the secretion shown in previous results.

Ethylene Glycol Modules


References
[1] S. Sulaiman, S. Yamato, E. Kanaya, J. Kim, Y. Koga, K. Takano, S. Kanaya. "Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach." Applied and Environment Microbiology, vol. 78 no. 5, pp. 1556-1562, March 2012.

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