Team:UANL Mty-Mexico/Project/detection

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iGEM UANL 2012


Detection


The genetic circuit used to build our arsenic biosensor is based on the Ars operon, which naturally occurs in E. coli. Ars operon detoxifies from arsenic, antimony and bismuth (Kaur et al. 1992). Briefly, the ArsR repressor normally inhibits the operon's expression; when arsenic is around, it binds the ArsR repressor therefore activating expression from pArsR (Figure 1a) (Wu & Rosen 1993). Our biosensor uses this regulatory mechanism, coupled to firefly's luciferase as a reporter gene, to quantify arsenic (Figure 1b).


Figure 1. a) Ars operon b) arsenic biosensor.

To achieve a better sensitivity, a 10 times more active mutant luciferase from Photinus pyralis (Fujii et al. 2007) will be used. 


Why luciferase?

Fluorescent proteins are dynamic reporter genes, useful for cellular assays but not proportional to the amount of activator. On the other hand, luciferase is an ideal reporter gene for experiments requiring high sensitivity, reproducibility and quantifiable results; i.e. the light emitted is proportional to luciferase expression (Devgan 2009).


Arsenic uptake

Arsenic is a toxic compound that is imported by passive diffusion through the membrane of E. coli. In order to increase the uptake of this metalloid, it is planned the expression of the aquaglycerol porin GlpF, which facilitates import of glycerol as well as arsenic and antimony (Figure 2)(Meng et al. 2004). Through this way it is expected to increase the biosensor sensibility for detection.


Figure 2.While the amount of arsenic imported by passive diffusion inside the cell is limited, we expect to obtain an increase in the uptake capacity of the component within the cell to capture via GlpF transport.

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