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Revision as of 23:29, 19 October 2016
Project Overview
Mechanism 1
Cell Free System
![Mechanism 2 overview diagram](https://static.igem.org/mediawiki/2016/8/86/T--Manchester--mechanism1_overview.png)
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 (E.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
![Mechanism 2 overview diagram](https://static.igem.org/mediawiki/2016/1/12/T--Manchester--mech2_overview.png)
The alc gene expression system is one of the most reliable chemically inducible gene switches for use in plants [2] and fungus [3]. While the alc gene switch has been shown to function in a variety of dicotyledonous plants including tobacco [4], Arabidopsis [5], poplar [6], potato [7], oilseed rape [7], and tomato [8], it has not been reported to be functional in monocotyledonous plants. 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 [8].
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.
- Caddick, M.X, et al. (1998). An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nature biotechnology , 16(2), pp. 177-80.
- Roslan, H.A., et al. (2001). Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana. The Plant Journal , 28(2), pp. 225-235.
- Filichkin S.A., Meilan R., Busov V.B., Ma C., Brunner A.M., Strauss S.H. (2006). Alcohol-inducible gene expression in transgenic Populus. Plant Cell 25, pp. 660–667.
- Sweetman J.P., Chu C., Qu N., Greenland A.J., Sonnewald U., Jepson I. (2002). Ethanol vapour is an efficient inducer of the alc gene expression system in model and crop plant species. Plant Physiology, 129, pp. 943–948.
- 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.