Difference between revisions of "Part:BBa K2753002"
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| − | <p>GPPS: Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS)<sup>[1]</sup>. IPP and DMAPP are supplied by either MVA pathway or MEP pathway or both depending on species. This parts originated from the species Abies Grandis, and it’s codon-optimized for the use in E. coli.</p> | + | <p>GPPS: Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS)<sup>[1]</sup>. IPP and DMAPP are supplied by either MVA pathway or MEP pathway or both depending on species. This parts originated from the species <em>Abies Grandis</em>, and it’s codon-optimized for the use in <em>E. coli</em>.</p> |
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<li>pUC20: ~500</li> | <li>pUC20: ~500</li> | ||
<li>pR6K: ~15</li> | <li>pR6K: ~15</li> | ||
| − | <li>pSC101: ~1<li> | + | <li>pSC101: ~1</li> |
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| − | <p>We co-expressed a heterologous yeast MVA pathway with the nine synthetic constructs in E. coli, obtaining nine E. coli strain potentially able to produce geraniol. Shake-flask fermentation is carried out with all strains along with the negative control strain containing only the MVA pathway. E. coli was incubated into 50ml LB supplemented with corresponding antibiotics, and the initial OD was adjusted to 0.01. When OD reached 1, 25uM of IPTG was added into the culture, and the culture was shaken at 37℃, 250rpm for 24h. Then 2ml of dodecane phase was added into 5ml of culture. The mixture was vortexed for 2min, and the organic phase was analyzed by gas chromatography to identify geraniol production. </p> | + | <p>We co-expressed a heterologous yeast MVA pathway with the nine synthetic constructs in <em>E. coli</em>, obtaining nine <em>E. coli</em> strain potentially able to produce geraniol. Shake-flask fermentation is carried out with all strains along with the negative control strain containing only the MVA pathway. <em>E. coli </em>was incubated into 50ml LB supplemented with corresponding antibiotics, and the initial OD<sub>600</sub> was adjusted to 0.01. When OD<sub>600</sub> reached 1, 25uM of IPTG was added into the culture, and the culture was shaken at 37℃, 250rpm for 24h. Then 2ml of dodecane phase was added into 5ml of culture. The mixture was vortexed for 2min, and the organic phase was analyzed by gas chromatography to identify geraniol production. </p> |
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Revision as of 11:25, 1 October 2018
pSB1C3-GPPS cds
GPPS: Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS)[1]. IPP and DMAPP are supplied by either MVA pathway or MEP pathway or both depending on species. This parts originated from the species Abies Grandis, and it’s codon-optimized for the use in E. coli.
Characterization
In our study, to investigate the optimal expression level of GPPS and GES for geraniol production, we assembled GPPS and obGES to make a geraniol-generating operon without a promoter(Part#), We employed three different promoters:
- pTALE sp1 (https://parts.igem.org/Part:BBa_K2753000), copy number-independent promoter of medium strength
- pTALE sp2 (https://parts.igem.org/Part:BBa_K2753001), copy number-independent promoter of low strength
- pTac (https://parts.igem.org/Part:BBa_K180000), IPTG inducible promoter
And three vectors of unique copy number:
- pUC20: ~500
- pR6K: ~15
- pSC101: ~1
In total, we obtained nine combinations of promoters and vectors which regulate the expression of GPPS with GES.
We co-expressed a heterologous yeast MVA pathway with the nine synthetic constructs in E. coli, obtaining nine E. coli strain potentially able to produce geraniol. Shake-flask fermentation is carried out with all strains along with the negative control strain containing only the MVA pathway. E. coli was incubated into 50ml LB supplemented with corresponding antibiotics, and the initial OD600 was adjusted to 0.01. When OD600 reached 1, 25uM of IPTG was added into the culture, and the culture was shaken at 37℃, 250rpm for 24h. Then 2ml of dodecane phase was added into 5ml of culture. The mixture was vortexed for 2min, and the organic phase was analyzed by gas chromatography to identify geraniol production.
The results indicate all combinations except for pTALEsp1 with high copy number generate significantly higher titer of geraniol compared to the negative control. Moreover, the combinations of pTac with high copy number and pTALE with lower copy number are more advantageous.
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]
Referece
[1]: Rai, A., Smita, S. S., Singh, A. K., Shanker, K., & Nagegowda, D. A. (2013). Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis. Molecular Plant, 6(5), 1531–1549. doi:10.1093/mp/sst058
[2]: Zebec, Z., Wilkes, J., Jervis, A. J., Scrutton, N. S., Takano, E., & Breitling, R. (n.d.). Towards synthesis of monoterpenes and derivatives using synthetic biology. Current Opinion in Chemical Biology, 34, 37–43. https://doi.org/10.1016/j.cbpa.2016.06.002