Part:BBa_K3595016

GPPS-MS regulated by pTac

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Usage and Biology

This part is a coding sequence after the promoter Ptac and RBS B0034. The enzyme geranyl pyrophosphate synthase can be translated under the induction of IPTG. We connected GPPS and MS with the promoter Ptac to express genes GPPS and MS on the plasmid pET28B. The constructed plasmid was transformed into the DH10b host cell to test its production of myrcene.

The structure of the plasmid pBR322-KanR-pTac-cysE and pBR322-KanR-pTac-cysE-mutant

Experimental Setup

• Genetic information of MS was described in the page Part: BBa_K35950015.
• Plasmid pTYT-GPPS-MS was transferred into E.coli DH10b host cell,respestively.
• Single colonies were selected from the experimental LB-agar plate , then inoculated into test-tube tubes with 4000 μL LB liquid medium with 4uL kanamycin for overnight growth at 37 °C and 200 rpm.
• DH10b was introduced in 2 ml M9 medium with 0.5 mM IPTG and 1% glucose, in 37 degree Celsius overnight. 20% dodecane (v/v) was added to collect the myrcene produced by DH10b.
• After the centrifugation (12000rpm for a minute), the dodecane layer was collected to test the production of myrcene.

Results

• DH10b with plasmids pMevT, pMBIS, and pTYT-GPPS-MS could produce myrcene, but in relatively low productivity compared to the previous research.

Detection result of the production of myrcene by the engineered bacteria. (A) GC-MS result of standard myrcene sample, blue arrow indicates the peak of myrcene. (B) GC-MS result of the culture solution of pMevT pMBIS pTYT-GPPS-MS transformed E.coli DH10b, blue arrow indicates the peak of myrcene. (C) The Calibration Line of myrcene. (D) The myrcene peak area and myrcene concentration of each group.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2573
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2438
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 741
  Illegal AgeI site found at 1919
  
• 1000
  COMPATIBLE WITH RFC[1000]