Difference between revisions of "Part:BBa K4417007"

 
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<partinfo>BBa_K4417007 short</partinfo>
 
<partinfo>BBa_K4417007 short</partinfo>
  
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<h1>Description</h1>
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This part is a novel cumate inducible promoter. This promoter was first synthesized by Claudia Schmidt-Dannert lab to induce the production of ''sf''GFP.
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<h1>Usage and Biology</h1>
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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<sub>veg</sub> with regulatory elements of the Pseudomonas putida, CymR repressor, and CuO operator sequence. CymR was expressed by a weak P<sub>xylR</sub> promoter, allowing the OFF and ON switch of an inducible ''sf''GFP reporter under the cumate-inducible promoter. In the absence of cumate, the CymR repressor will bind to the CuO operator and block ''sf''GFP production from the P<sub>veg</sub> promoter. With cumate, the CymR will be released from the CuO operator; therefore, the ''sf''GFP expression will be switched on.
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[[File:Zjy21.png|600px|thumb|center|'''Figure 1:''' Inducible cumate promoter expression system.]]
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Research literature indicates three important features of this promoter:
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* Higher protein expression levels in ''Bacillus'' compared to other reported inducible ''Bacillus'' expression systems.
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* Also inducible in ''E. coli''.
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* Tuneable protein expression levels by varying cumate concentration.
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<h1>Method</h1>
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Cumate is non-toxic to the host. In our experiment, we tried to induce the ''sf''GFP expression in ''E. coli'' with different cumate concentrations (10-100 μM).
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<h1>Characterization</h1>
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''''Cumate testing''''
  
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===Usage and Biology===
 
  
 
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Revision as of 10:28, 12 October 2022


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 Pveg with regulatory elements of the Pseudomonas putida, CymR repressor, and CuO operator sequence. CymR was expressed by a weak PxylR 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 Pveg 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]