Difference between revisions of "Part:BBa K3052001"
m (→‎Characterization)
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===Background===
 
===Background===
  
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In our study, we aim to achieve limonene and linalool synthesis in <it>E.coli</it> DH5<it>α</it>. According to 2018 GreatBay_China’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.
+
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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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:  
  
−
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.  
+
1. ([parts.igem.org/Part:BBa_K3052001 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.  
  
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2. (BBa_K3052004, BBa_K3052010) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in <it>E. coli</it>.
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2. ([parts.igem.org/Part:BBa_K3052004 BBa_K3052004], BBa_K3052010) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in <it>E. coli</it>.

Revision as of 01:08, 16 October 2019


(4S)-limonene synthase

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).

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Background

In our study, we aim to achieve limonene and linalool synthesis in E.coli DH5 α. 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. ([parts.igem.org/Part:BBa_K3052001 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. ([parts.igem.org/Part:BBa_K3052004 BBa_K3052004], BBa_K3052010) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in <it>E. coli</it>.