Part:BBa_K4417007

CuO Cumate Inducible System

Description

This part is a novel cumate inducible promoter. This promoter was first synthesized by Claudia Schmidt-Dannert lab to induce the production of sfGFP.

Usage and Biology

Cumate, as an inducer, is non-toxic at working concentrations, less expensive than IPTG, and not dependent on a carbon source, making it a cost-effective and efficient method for inducing protein expression in large-scale industrial production. p-isopropyl benzoate (cumate) inducible gene expression system was constructed by combining the strong constitutive Bacillus promoter P_veg with regulatory elements of the Pseudomonas putida, CymR repressor, and CuO operator sequence. CymR was expressed by a weak P_xylR promoter, allowing the OFF and ON switch of an inducible sfGFP reporter under the cumate-inducible promoter. In the absence of cumate, the CymR repressor will bind to the CuO operator and block sfGFP production from the P_veg promoter. With cumate, the CymR will be released from the CuO operator; therefore, the sfGFP expression will be switched on.

Figure 1: Inducible cumate promoter expression system.

Research literature indicates three important features of this promoter:

• Higher protein expression levels in Bacillus compared to other reported inducible Bacillus expression systems.
• Also inducible in E. coli.
• Tuneable protein expression levels by varying cumate concentration.

Method

Cumate is non-toxic to the host. In our experiment, we tried to induce the sfGFP expression in E. coli with different cumate concentrations (10-100 μM).

Characterization

'Cumate testing'

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]