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Team:UC Davis/Project/Catalyst
From 2012.igem.org
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We have created several modules and biobrick parts for the degradation and utilization of PET. The PET degradation can be seen below. | We have created several modules and biobrick parts for the degradation and utilization of PET. The PET degradation can be seen below. | ||
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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). | 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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<center><img src="https://static.igem.org/mediawiki/2012/6/62/UCDavisLCCutinase_Process.png"></center> | <center><img src="https://static.igem.org/mediawiki/2012/6/62/UCDavisLCCutinase_Process.png"></center> | ||
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| + | We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and labeled it <a href="http://partsregistry.org/Part:BBa_K936014">Bba_K936014</a>. It has been placed the following construct where it is promoted by a pBad variant Bba_K206000. | ||
| + | <center><br><img src="https://static.igem.org/mediawiki/2012/2/28/UCDavisNew_pBAD_B34_Cutinase.png"></center> | ||
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The pelB leader sequence on the cutinase gene directs 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. | The pelB leader sequence on the cutinase gene directs 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. | ||
Revision as of 21:41, 22 September 2012
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Modules
LC-Cutinase and Initial PET Degradation
We had the LC-Cutinase gene synthesized with a pelB leader sequence and a 6-his tag and labeled it Bba_K936014. It has been placed the following construct where it is promoted by a pBad variant Bba_K206000.
The pelB leader sequence on the cutinase gene directs 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. The inclusion of a his-tag allows us to purify the cutinase protein and identify where it is after it is produced. We have designed and conducted an experiment to determine how much of the protein is being secreted and how much is remaining inside of the cell, the results of which can be found here (link coming soon).
