Difference between revisions of "Part:BBa K3052004"

 
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<partinfo>BBa_K3052004 short</partinfo>
 
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The linalool synthase is used to convert geranyl pyrophosphate(GPP) to linalool, which has aroma of lavendar.
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The linalool synthase is used to convert geranyl pyrophosphate (GPP) to linalool, which has aroma of lavendar.
  
 
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<partinfo>BBa_K3052004 parameters</partinfo>
 
<partinfo>BBa_K3052004 parameters</partinfo>
 
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==Characterization==
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===Background===
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In our study, we aim to achieve limonene and linalool synthesis in <i>E.coli</i> DH5<i>α</i>. According to 2018 GreatBay_China team’s experience, no target product was detected using gas chromatography when carrying out shake-flask fermentation with this strain induced by 25uM IPTG for 24 hours due to the lack of endogenous MVA pathways wherein GPP is produced. Thus we decided to co-express an MVA pathway. 2018 GreatBay_China generously gave us one plasmid pJBEI6409, which contains a MVA pathway in addition to an GPPs-LS operon. We reconstructed this plasmid and get a plasmid only contains a MVA pathway.
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<div>[[File:T--XJTU-CHINA--MVA pathway.png |700px|thumb|center|<b>Figure 1:</b> MVA pathway]]</div>
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<div>[[File:T--XJTU-CHINA--GPP_production_and_synthesis.png |700px|thumb|center|<b>Figure 2:</b> GPP production and synthesis of linalool and limonene]]</div>
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We have used MVA synthesis pathway which is common in plants. Since limonene and linalool have the same synthetic precursor GPP, we have divided the synthesis pathway into two parts:
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1. (<partinfo>BBa_K3052001</partinfo>) IPTG inducible precursor circuit which contains eight enzymes of MVA pathway enable conversion from AcCoA to GPP: atoB, HMGS, HMGR, MK, PMK, PMD, idi, trGPPs.
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2. (<partinfo>BBa_K3052004</partinfo>, <partinfo>BBa_K3052010</partinfo>) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in <i>E. coli</i>.
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 +
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===Results===
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 +
For validation of these parts, we did both RT-PCR and SDS-PAGE to detect the expression, Gas Chromatography to detect limonene and linalool production. Click to see our [http://2019.igem.org/Team:XJTU-CHINA protocol].
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====RT-PCR====
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<div>[[File:T--XJTU-CHINA--Relative_mRNA_level.png |700px|thumb|center|<b>Figure 3:</b> Relative mRNA level of 3 experimental group ( pGPP’s mRNA level is defined 1)]]</div>
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====SDS-PAGE====
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<div>[[File:T--XJTU-CHINA--3_experimental_group_and_1_control_group_pGPP.png |700px|thumb|center|<b>Figure 4:</b> 3 experimental group and 1 control group pGPP (+: with IPTG induction / -: without IPTG induction)]]</div>

Revision as of 01:46, 16 October 2019


(S)-linalool synthase: DHJ

The linalool synthase is used to convert geranyl pyrophosphate (GPP) to linalool, which has aroma of lavendar.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 490
    Illegal PstI site found at 571
    Illegal PstI site found at 994
    Illegal PstI site found at 1060
    Illegal PstI site found at 1150
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 490
    Illegal PstI site found at 571
    Illegal PstI site found at 994
    Illegal PstI site found at 1060
    Illegal PstI site found at 1150
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 490
    Illegal PstI site found at 571
    Illegal PstI site found at 994
    Illegal PstI site found at 1060
    Illegal PstI site found at 1150
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 490
    Illegal PstI site found at 571
    Illegal PstI site found at 994
    Illegal PstI site found at 1060
    Illegal PstI site found at 1150
    Illegal NgoMIV site found at 21
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Background

In our study, we aim to achieve limonene and linalool synthesis in E.coli DH5α. According to 2018 GreatBay_China team’s experience, no target product was detected using gas chromatography when carrying out shake-flask fermentation with this strain induced by 25uM IPTG for 24 hours due to the lack of endogenous MVA pathways wherein GPP is produced. Thus we decided to co-express an MVA pathway. 2018 GreatBay_China generously gave us one plasmid pJBEI6409, which contains a MVA pathway in addition to an GPPs-LS operon. We reconstructed this plasmid and get a plasmid only contains a MVA pathway.


Figure 1: MVA pathway
Figure 2: GPP production and synthesis of linalool and limonene

We have used MVA synthesis pathway which is common in plants. Since limonene and linalool have the same synthetic precursor GPP, we have divided the synthesis pathway into two parts:

1. (BBa_K3052001) IPTG inducible precursor circuit which contains eight enzymes of MVA pathway enable conversion from AcCoA to GPP: atoB, HMGS, HMGR, MK, PMK, PMD, idi, trGPPs.

2. (BBa_K3052004, BBa_K3052010) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in E. coli.


Results

For validation of these parts, we did both RT-PCR and SDS-PAGE to detect the expression, Gas Chromatography to detect limonene and linalool production. Click to see our [http://2019.igem.org/Team:XJTU-CHINA protocol].


RT-PCR

Figure 3: Relative mRNA level of 3 experimental group ( pGPP’s mRNA level is defined 1)


SDS-PAGE

Figure 4: 3 experimental group and 1 control group pGPP (+: with IPTG induction / -: without IPTG induction)