Difference between revisions of "Part:BBa K3595017"
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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. | 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. | ||
| − | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here--> |
| − | + | =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 <i>E.coli DH10b </i> host cell with plasimd pMevT, pMBIS to test its production of myrcene. | ||
| + | [[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 ]] | ||
| + | [[File:T--GZ_HFI--GPPS-MS.png|600px|thumb|center|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 <i>E.coli DH10b </i> 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== | ||
| + | * <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. | ||
| + | [[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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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K3595017 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3595017 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | <!-- Add more about the biology of this part here--> | ||
| + | =Reference= | ||
| + | Kim EM, et al. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli. J Agric Food Chem 2015,13;63(18):4606-12.<br> | ||
| + | Martin VJJ, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nat Biotechnol 2003;21(7):796-802.<br> | ||
| + | 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> | ||
Latest revision as of 18:16, 27 October 2020
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.
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:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2573
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2438
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 741
Illegal AgeI site found at 1919 - 1000COMPATIBLE 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.