Part:BBa_K3595017

GPPS-MS regulated by pTac

use pTac promotor (BBa_K3595003),strong RBS (BBa_B0034) , RiboJ(BBa_K2615996), coding sequence GPPS(BBa_K3595016), coding sequence MS(BBa_K3595015) and terminator(BBa_B0010) ,will generate GGP and MS in case of IPTG.

Usage and Biology

This composite part can be expressed downstream of the promoter pTac in the presence of IPTG. We constructed plasmids pBR322-KanR-pTac-GPPS-MS. The constructed plasmid was cotransferred into E.coli DH10b host cell with plasimd pMevT, pMBIS to test its production of myrcene.

The metabolism pathway of myrcene. (A) The MevT pathway. (B) MBIS Pathway and genes GPPS and MS

The structure of the plasmid pBR322-KanR-pTac-GPPS-MS

Experimental Setup

• Genetic information of MS,GPPS was described in the page Part:BBa_K3595015, Part:BBa_K3595014,respectively.
• Plasmid pBR322-KanR-pTac-GPPS-MS,pMevT and pMBIS were cotransferred into the E.coli DH10b competent cell .
• Single colonies were selected from the experimental LB-agar plate with antibiotics, then inoculated into test-tube tubes with

2 ml M9 medium with 0.1 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

• E.coli 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]

Reference

Kim EM, et al. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli. J Agric Food Chem 2015,13;63(18):4606-12.
Martin VJJ, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nat Biotechnol 2003;21(7):796-802.
Nakatani T, et al. Enhancement of thioredoxin: glutaredoxin- mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. Microb Cell Fact 2012;11:62.