Team:ETH Zurich/Biology/Validation

AlcR sensor

Design of the AlcR testsystem AlcR gets produced upon addition of anhydrotetracycline. A 6x His-tag was added to the C-terminal end of AlcR to test the expression of alcR.

As described in the Smoke detectors section we designed an artificial AlcR-dependent promoter which gets repressed upon binding of AlcR to acetaldehyde. Fortesting of our design we created a system with a superfolded GFP output in order to characterize the gene expression with different levels of AlcR production and acetaldehyde induction.

The system work the following way: A) In normal medium conditions the TetR transcription factor is produced and binds to its cognate promoter, thus inhibiting the expression of alcR. In this case no AlcR is present and GFP is produced. B) Upon addition of increasing amounts of anhydrotetracycline, TetR gets released from the promoter and AlcR is produced. The amount of AlcR should increase with increasing amounts of anhydrotetracycline. C) By addition of acetaldehyde AlcR binds to the alc-promoter and the GFP response decreases.

[[File:Test Palc1.png|300px|left|thumb|] [[File:Test Palc2.png|300px|right|thumb|]

Expression of AlcR in E. coli

Given that codon usage is not the same in E. coli and Aspergillus nidulans, we analyzed codon usage of AlcR. We obtained a Codon adaptation index (CAI) of 0.7 for the Aspergillus nidulans protein in E. coli, especially at the beginning of the gene rare codons were included. To get a fully E. coli-optimized gene, we ordered the gene codon optimized. To make some first test, we exchanged the rare codons at the beginning of the Aspergillus nidulans protein with PCR. With these protein we made same first Test of the expression of AlcR.

To monitor the expression of AlcR a His-tag was introduced in the test system.

western blot

Repression test of AlcR

To test our designed AlcR-promotors the following system was designed (see Figure). In present of acetaldeyhde AlcR binds to the AlcR operon and inhibits the expression of GFP. For a better signal assam gfp was used.

Design of our testsystem.

results

XylR sensor

Xylene degradation

Bandpass

References

[1] [http://www.ncbi.nlm.nih.gov/pubmed/11550794 Beatrice Felenbok, Michel Flipphi and Igor Nikolaev: Ethanol Catabolism in Aspergillus nidulans: A Model System for Studying Gene Regulation, Progress in Nucleic Acid Research and Molecular Biology, 69: 149-204]