View source for Part:BBa K3052001


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<partinfo>BBa_K3052001 short</partinfo>

The limonene synthase (LS) sequence used throughout our project which converts GPP to limonene is an E. coli codon-optimized version of a truncated sequence from M. spicata previously described(Hyatt et al., 2007). 

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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K3052001 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K3052001 parameters</partinfo>
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==Characterization==

===Background===

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.


<div>[[File:T--XJTU-CHINA--MVA pathway.png |700px|thumb|center|<b>Figure 1:</b> MVA pathway]]</div>
<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>

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. (<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. 

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>.


===Results===

For validation of this parts, limonene synthase CS under the control of Ptrc were ligated to pSB1K3 to validate the activity and function. And the pGPP plasmid and CS-harboring pCS was co-transformed into E. coli DH5α for limonene synthesis. we did both RT-qPCR and SDS-PAGE to detect the expression, and Gas Chromatography (GC)to detect limonene and linalool production. Click to see our [http://2019.igem.org/Team:XJTU-CHINA protocol].


====RT-PCR====

<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>


====SDS-PAGE====

<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>