Team:ETH Zurich/Biology/Validation

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Revision as of 09:22, 21 September 2011

Can you feel the smoke tonight?

Validation

Experimental setups...

AlcR sensor

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]

@@ Line 22: / Line 22: @@
 == 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.
+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 a few very 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.
+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.