Difference between revisions of "Part:BBa K3052010"
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<partinfo>BBa_K3052010 short</partinfo>
 
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==Characterization==
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==Validation==
  
 
===Background===
 
===Background===
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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--project3.png |700px|thumb|center|<b>Figure 1:</b> (A)MVA pathway. (B)Synthesis of limonene and linalool]]</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:  
 
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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===Results===
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===Characterization===
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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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Precursor Circuit converts carbon sources like glucose to geranyl pyrophosphate(GPP), ready to be catalyzed into limonene and linalool. We constructed the circuit by modification of pJBEI6409. The 11523 bp-length vector is constructed based on pSB1C3 backbone and each operon is under the regulation of Ptrc, and the digestion verification result of two endonucleases is as follows.
  
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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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<div>[[File:T--XJTU-CHINA--pGPP.png |700px|thumb|center|<b>Figure 2:</b>Endonuclease digestion verification of precursor circuit]]</div>
  
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Since the product of this circuit, GPP is unstable to be detected via HPLC etc., we performed SDS-PAGE analysis to compare the expression level of key enzyme--GGPs with and without IPTG induction to test if this circuit works properly, and the result is shown below. Click to see our protocol.
  
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====SDS-PAGE====
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<div>[[File:T--XJTU-CHINA--PAGE-pGPP.png |700px|thumb|center|<b>Figure 3:</b>SDS-PAGE result of precursor circuit (+:with IPTG induction/-: without IPTG induction)]]</div>
  
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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>
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The results of SDS-PAG clearly demonstrate the high expression of the GPPs in the precursor circuit, indicating its functionality and eventual GPP synthesis.

Revision as of 17:35, 21 October 2019

Precursor Circuit -- a series of enzymes used for GPP production

This circuit contains eight enzymes in plant's MVA pathway to produce geranyl pyrophosphate(GPP),a precursor to various metabolites unique in plants like linalool and limonene. All of the genes in this circuit are inducible by IPTG since all of them are under control of Ptrc or Plac.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1557
    Illegal XbaI site found at 8869
    Illegal SpeI site found at 10020
    Illegal PstI site found at 4253
    Illegal PstI site found at 6546
    Illegal PstI site found at 6837
    Illegal PstI site found at 7196
    Illegal PstI site found at 11005
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1557
    Illegal NheI site found at 3467
    Illegal SpeI site found at 10020
    Illegal PstI site found at 4253
    Illegal PstI site found at 6546
    Illegal PstI site found at 6837
    Illegal PstI site found at 7196
    Illegal PstI site found at 11005
    Illegal NotI site found at 7688
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1557
    Illegal BglII site found at 1566
    Illegal XhoI site found at 11386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1557
    Illegal XbaI site found at 8869
    Illegal SpeI site found at 10020
    Illegal PstI site found at 4253
    Illegal PstI site found at 6546
    Illegal PstI site found at 6837
    Illegal PstI site found at 7196
    Illegal PstI site found at 11005
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1557
    Illegal XbaI site found at 8869
    Illegal SpeI site found at 10020
    Illegal PstI site found at 4253
    Illegal PstI site found at 6546
    Illegal PstI site found at 6837
    Illegal PstI site found at 7196
    Illegal PstI site found at 11005
    Illegal NgoMIV site found at 1443
    Illegal NgoMIV site found at 5780
    Illegal NgoMIV site found at 7726
    Illegal AgeI site found at 8179
  • 1000
    COMPATIBLE WITH RFC[1000]


Validation

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: (A)MVA pathway. (B)Synthesis of limonene and linalool

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.


Characterization

Precursor Circuit converts carbon sources like glucose to geranyl pyrophosphate(GPP), ready to be catalyzed into limonene and linalool. We constructed the circuit by modification of pJBEI6409. The 11523 bp-length vector is constructed based on pSB1C3 backbone and each operon is under the regulation of Ptrc, and the digestion verification result of two endonucleases is as follows.

Figure 2:Endonuclease digestion verification of precursor circuit

Since the product of this circuit, GPP is unstable to be detected via HPLC etc., we performed SDS-PAGE analysis to compare the expression level of key enzyme--GGPs with and without IPTG induction to test if this circuit works properly, and the result is shown below. Click to see our protocol.

Figure 3:SDS-PAGE result of precursor circuit (+:with IPTG induction/-: without IPTG induction)

The results of SDS-PAG clearly demonstrate the high expression of the GPPs in the precursor circuit, indicating its functionality and eventual GPP synthesis.