Difference between revisions of "Team:Manchester/Description"
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<img style="width:60%;margin:auto" src="https://static.igem.org/mediawiki/2016/8/86/T--Manchester--mechanism1_overview.png" alt="Mechanism 2 overview diagram" /> | <img style="width:60%;margin:auto" src="https://static.igem.org/mediawiki/2016/8/86/T--Manchester--mechanism1_overview.png" alt="Mechanism 2 overview diagram" /> | ||
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| + | <p style="font-size:20px;text-align:left">Enzymatic colorimetric assays are used to determine the concentration of a chemical in a solution by the conversion of a chromogen substrate into a coloured product. We have engineered <i>Escherichia coli</i> BL21 (DE3) strain to express AOx from <i>Pichia pastoris</i> that will then be used in a cell-free colorimetric system. This method involves the usage of alcohol oxidase (AOx) to oxidise ethanol producing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) as a by-product. H2O2 is used as an oxidising agent by horseradish peroxidase (HRP) to convert ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) to produce the colour change <sup>[1]</sup>. | ||
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| − | <p style="font-size: 20px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants and fungus. This system relies on the ability of AlcR, a transcription factor, to bind to its target <i>alcA</i> promoter (alcA<sup>P</sup>). Based on this, we have engineered <i>Escherichia coli</i> K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to alcA<sup>P</sup> in the presence of ethanol <sup>[ | + | <p style="font-size: 20px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants and fungus. This system relies on the ability of AlcR, a transcription factor, to bind to its target <i>alcA</i> promoter (alcA<sup>P</sup>). Based on this, we have engineered <i>Escherichia coli</i> K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to alcA<sup>P</sup> in the presence of ethanol <sup>[2]</sup>. |
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| + | <li>Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M., & Fonseca, L. P. (2005). Ethanol biosensors based on alcohol oxidase. <i>Biosensors and Bioelectronics</i>,21(2), 235-247. | ||
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<li>Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997) ‘The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans’, <i>Journal of Biological Chemistry</i>, 272(36), pp. 22859–22865. | <li>Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997) ‘The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans’, <i>Journal of Biological Chemistry</i>, 272(36), pp. 22859–22865. | ||
</li> | </li> | ||
Revision as of 20:15, 28 September 2016
Project Overview
Mechanism 1
Cell Free Mechanism
Enzymatic colorimetric assays are used to determine the concentration of a chemical in a solution by the conversion of a chromogen substrate into a coloured product. We have engineered Escherichia coli BL21 (DE3) strain to express AOx from Pichia pastoris that will then be used in a cell-free colorimetric system. This method involves the usage of alcohol oxidase (AOx) to oxidise ethanol producing hydrogen peroxide (H2O2) as a by-product. H2O2 is used as an oxidising agent by horseradish peroxidase (HRP) to convert ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) to produce the colour change [1].
Mechanism 2
Inducible Gene Switch
The alc gene expression system is one of the most reliable chemically inducible gene switches for use in plants and fungus. This system relies on the ability of AlcR, a transcription factor, to bind to its target alcA promoter (alcAP). Based on this, we have engineered Escherichia coli K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to alcAP in the presence of ethanol [2].
Reference
- Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M., & Fonseca, L. P. (2005). Ethanol biosensors based on alcohol oxidase. Biosensors and Bioelectronics,21(2), 235-247.
- Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997) ‘The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans’, Journal of Biological Chemistry, 272(36), pp. 22859–22865.
