BBa_K875003

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Description

This composite produces constitutively the CymR regulator protein, that binds the Cumate Operator repressing the transcription from the promoter.

Assembly

The CymR gene was obtained by synthesis and then was assembled with other BioBricks.

Results

CymR expression was confirmed by Western Blot analysis. Our CymR has a SV5 tag at the C-terminus in order to be detected by an anti-SV5 Ab. Its functionality was confirmed through the repression of T5 Cumate Operator-GFP.

The validation of the repression of the protein CymR on the T5 cumate operator was verified through the creation of a plasmid containing a single copy of CymR and the T5 cumate operator upstream the GFP (I13504). We saw that the expression of the GFP was completely repressed in absence of p-cumate but it was active in presence of p-cumate.

Post European-Jamboree

Cuminaldehyde activation of cumate switch

Up to now all our tests were based on p-cumate according to the literature

(Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains Young J. Choi, Lyne Morel, Teffanie Le François Denis Bourque, Lucie Bourget, Denis Groleau, Bernard Massie and Carlos B. Míguez Applied and Environmental Microbiology, Aug. 2010, p. 5058–5066 Vol.76 doi:10.1128/AEM.00413-10)

. But since it is known that the most abundant component in the cumin spice is the cuminaldehyde, we later decided to test also its activity in the regulation of the cumate switch. We confirmed that also the cuminaldehyde was able to activate the cumate switch, although in higher concentrations.