Difference between revisions of "Part:BBa K3052001"
(→Results) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K3052001 short</partinfo> | <partinfo>BBa_K3052001 short</partinfo> | ||
| − | The limonene synthase ( | + | The limonene synthase (CS) 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). |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
| Line 22: | Line 22: | ||
===Background=== | ===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 | + | 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 conduct shake-flask fermentation with this strain induced by 25uM IPTG for 24 hours due to the lack of endogenous MVA pathways. 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-CS operon. We reconstructed this plasmid to generate pGPP and pCS plasmids. |
Revision as of 14:28, 21 October 2019
(4S)-limonene synthase
The limonene synthase (CS) 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 conduct shake-flask fermentation with this strain induced by 25uM IPTG for 24 hours due to the lack of endogenous MVA pathways. 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-CS operon. We reconstructed this plasmid to generate pGPP and pCS plasmids.
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 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
