Difference between revisions of "Team:Manchester/Description"
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| − | <p style="font-size:18px;text-align:left">Enzymatic colourimetric 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 introduced a plasmid expressing recombinant Alcohol Oxidase 1 (<a href="http://parts.igem.org/Part:BBa_K2092000" target="_blank">AOx</a>) from <i>Pichia pastoris</i> into <i>Escherichia coli | + | <p style="font-size:18px;text-align:left">Enzymatic colourimetric 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 introduced a plasmid expressing recombinant Alcohol Oxidase 1 (<a href="http://parts.igem.org/Part:BBa_K2092000" target="_blank">AOx</a>) from <i>Pichia pastoris</i> into <i>Escherichia coli</i> BL21 (DE3) strain that will then be used in the cell-free colorimetric system. This method involves the usage of AOx to oxidise ethanol, producing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) as a by-product. H<sub>2</sub>O<sub>2</sub> 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: 18px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants <sup>[2]</sup> and fungus <sup>[3]</sup> | + | <p style="font-size: 18px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants <sup>[2]</sup> and fungus <sup>[3]</sup>. |
| − | This system relies on the ability of <a href="http://parts.igem.org/Part:BBa_K2092001" target="_blank"> AlcR</a>, an alcohol-activated transcription factor, to bind to its target <i>alcA</i> promoter (<a href="http://parts.igem.org/Part:BBa_K2092002" target="_blank"> | + | This system relies on the ability of <a href="http://parts.igem.org/Part:BBa_K2092001" target="_blank"> AlcR</a>, an alcohol-activated transcription factor, to bind to its target <i>alcA</i> promoter (<a href="http://parts.igem.org/Part:BBa_K2092002" target="_blank">P<i>alc</i>A</a>). Based on this, we have engineered <i>E. coli</i> K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to the native P<i>alc</i>A and variant of P<i>alc</i>A (<a href="http://parts.igem.org/Part:BBa_K2092003" target="_blank"> P<i>alc</i>A(var)</a>) in the presence of ethanol <sup>[4]</sup>. |
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<li style="text-align: left;;"> 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 style="text-align: left;;"> 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. | ||
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<li style="text-align: left;;"> Garoosi, A.G., Salter,M.G. , Caddick ,X.M and Tomsett, M.B. (2004). Characterization of the ethanol-inducible <i>alc</i> gene expression system in tomato. <i>Journal of experimental Botany</i>, 46 (416), pp. 1635-1642. | <li style="text-align: left;;"> Garoosi, A.G., Salter,M.G. , Caddick ,X.M and Tomsett, M.B. (2004). Characterization of the ethanol-inducible <i>alc</i> gene expression system in tomato. <i>Journal of experimental Botany</i>, 46 (416), pp. 1635-1642. | ||
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| + | <a class="projectlink" href="https://2016.igem.org/Team:Manchester"><< Main Page</a> | ||
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Latest revision as of 14:39, 24 November 2016
Project Overview
Mechanism 1
Cell Free System
Enzymatic colourimetric 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 introduced a plasmid expressing recombinant Alcohol Oxidase 1 (AOx) from Pichia pastoris into Escherichia coli BL21 (DE3) strain that will then be used in the cell-free colorimetric system. This method involves the usage of 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 [2] and fungus [3]. This system relies on the ability of AlcR, an alcohol-activated transcription factor, to bind to its target alcA promoter (PalcA). Based on this, we have engineered E. coli K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to the native PalcA and variant of PalcA ( PalcA(var)) in the presence of ethanol [4].
References
- 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.
- Plants: Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.
- 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.
- Garoosi, A.G., Salter,M.G. , Caddick ,X.M and Tomsett, M.B. (2004). Characterization of the ethanol-inducible alc gene expression system in tomato. Journal of experimental Botany, 46 (416), pp. 1635-1642.
