Team:ETH Zurich/Biology/Cloning

From 2011.igem.org

Revision as of 11:54, 5 September 2011

Can you feel the smoke tonight?

Used parts from registry

• BBa R0040 P_Tet
• BBa R0051 λ_P
• BBa R0010 P_lac
• BBa R0061 P_lux
• BBa J23100 P_const
• BBa C0061 LuxI_LVA
• BBa C0040 TetR_LVA
• BBa C0051 CI
• BBa C0062 LuxR
• BBa B0015 Double Terminator

Synthesized parts

• LacI_M1
• AlcR (codon optimized)

Design of a AlcR/acetaldeyhde repressed promotor P_AlcR

For the promotor P_AlcR the operator site of AlcR-inducer from apergillus nidulans was placed between the -10 and -35 region of the strong promotor. While acetaldeyhde is bind to AlcR, the complex will bind to the operon and block the binding of the RNA-polymerase.

Design of the promotor for the AlcR-system, the operon sequence is designed between the -10 and the -35 region (blue) of a strong promotor with inverted and direct-repeats.

Design of the test-system

To test our designed AlcR-promotors the following system was designed (see Figure). To induce the expression of AlcR a tetracycline induced promotor was used. To monitor the expression of AlcR a His-tag was introduced. 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.

Characterization of LacI_M1