"
Team:UC Davis/Project/Catalyst
From 2012.igem.org
(Difference between revisions)
| Line 409: | Line 409: | ||
<article> | <article> | ||
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). | ||
| - | <br> | + | <br><img src="https://static.igem.org/mediawiki/2012/6/62/UCDavisLCCutinase_Process.png"> |
<br> | <br> | ||
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. | 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. | ||
Revision as of 23:11, 28 August 2012
Modules
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)
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).
