Difference between revisions of "Part:BBa K3595016"
 
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=Usage and Biology=
 
=Usage and Biology=
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This part is a coding sequence after the promoter Ptac and RBS [[Part:B0034|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 . The constructed plasmid was transformed into the DH10b host cell to test its production of myrcene.
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This part，[[Part:BBa_K2615996]]，[[Part:BBa_B0034]] and [[Part:BBa_K3595015]] can be combined to form a composite part，which
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[[File:T--GZ_HFI--cysE.png|600px|thumb|center|The structure of the plasmid pBR322-KanR-pTac-cysE and pBR322-KanR-pTac-cysE-mutant ]]
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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 <i>E.coli DH10b </i> host cell with plasimd pMevT, pMBIS to test its production of myrcene.
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[[File:T--GZ_HFI--GPPS-Design.png|600px|thumb|center|The metabolism pathway of myrcene. (A) The MevT pathway. (B) MBIS Pathway and genes GPPS and MS ]]
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[[File:T--GZ_HFI--GPPS-MS.png|600px|thumb|center|The structure of the plasmid pBR322-KanR-pTac-GPPS-MS ]]
 
==Experimental Setup==
 
==Experimental Setup==
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*Genetic information of MS was described in the page [[Part: BBa_K35950015]].
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*Genetic information of MS,GPPS was described in the page [[Part:BBa_K3595015]], [[Part:BBa_K3595016]],respectively.
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*Plasmid pTYT-GPPS-MS was transferred into <i>E.coli DH10b </i> host cell,respestively.  
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*Plasmid pBR322-KanR-pTac-GPPS-MS,pMevT and pMBIS  were cotransferred into the <i>E.coli DH10b </i> competent cell .  
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*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.
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*Single colonies were selected from the experimental LB-agar plate with antibiotics, then inoculated into test-tube tubes with   
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*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.
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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.
 
*After the centrifugation (12000rpm for a minute), the dodecane layer was collected to test the production of myrcene.
 
==Results==
 
==Results==
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*DH10b with plasmids pMevT, pMBIS, and pTYT-GPPS-MS could produce myrcene, but in relatively low productivity compared to the previous research.  
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* <i>E.coli DH10b </i>  with plasmids pMevT, pMBIS, and pTYT-GPPS-MS could produce myrcene, but in relatively low productivity compared to the previous research.
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[[File:T--GZ_HFI--MS.png|600px|thumb|center|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 pBR322-KanR-pTac-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. ]]
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[[File:T--GZ_HFI--MS.png|600px|thumb|center|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 ]]
  
  
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<partinfo>BBa_K3595016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3595016 SequenceAndFeatures</partinfo>
  
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=Reference=
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Kim EM, et al. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli. J Agric Food Chem 2015,13;63(18):4606-12.<br>
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Martin VJJ, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nat Biotechnol 2003;21(7):796-802.<br>
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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.<br>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 18:21, 27 October 2020


Coding gene GPPS

This gene is derived from Abies grandies. This gene can encode the enzyme geranyl pyrophosphate synthase, which is used to catalyzed the reaction of IPP and DMAPP to form geranyl diphosphate, the precursor of monoterpene. In our project, the geen GPPS is constructed into plasmid to form pBR322-KanR-pTac-GPPS-MS.

Usage and Biology

This part，Part:BBa_K2615996，Part:BBa_B0034 and Part:BBa_K3595015 can be combined to form a composite part，which 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_K3595016,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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 640
  • 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.