Difference between revisions of "Part:BBa K2753003"
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===Characterization===
 
===Characterization===
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<p>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: </p><br>
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<p> In our study, we aim to achieve geraniol synthesis in E. coli, so at the beginning, we expressed a geraniol synthesis operon (https://parts.igem.org/Parts:BBa_K2753016) containing an Abies Grandis geranyl pyrophosphate synthase (https://parts.igem.org/Parts:BBa_K2753002) and an Ocimum basilicum geraniol synthase (https://parts.igem.org/Parts:BBa_K2753003) regulated by a pTac promoter in E. coli. Assuming that higher expression leads to higher yield, the operon was assembled on a high copy vector pUC20 whose copy number is approximately 500 per cell. However, when carrying out shake-flask fermentation with this strain, after induced by 25uM IPTG for 24 hours, no geraniol production was detected using gas chromatography. (see detailed methods in our notebook LINK)
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We infer that the failure to produce geraniol was due to the lack of substrates IPP and DMAPP. Thus we decided to co-express an MVA pathway in addition to the geraniol synthesis operon. The pathway can either be<sup>[5]</sup> added by cotransformation of a plasmid containing only the MVA pathway and the other plasmid with only GPPS and GES, or by constructing an all-in-one plasmid containing the MVA pathway, GPPS, and GES. We obtained a plasmid pMVA-only which was a gift from Taek Soon Lee, and have added GPPS and GES onto pMVA-only to acquire a pMVA-GPPS-GES. </p><br>
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<li>pTALE sp1 (https://parts.igem.org/Part:BBa_K2753000), copy number-independent promoter of medium strength</li>
 
<li>pTALE sp1 (https://parts.igem.org/Part:BBa_K2753000), copy number-independent promoter of medium strength</li>
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<p>In total, we obtained nine combinations of promoters and vectors which regulate the expression of GPPS with GES. </p>
 
<p>In total, we obtained nine combinations of promoters and vectors which regulate the expression of GPPS with GES. </p>
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<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 30℃, 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 30℃, 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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===Reference===
 
===Reference===
 
[1]:  Chen, F., Li, W., Jiang, L., Pu, X., Yang, Y., & Zhang, G. (2016). Functional characterization of a geraniol synthase ‑ encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. https://doi.org/10.1007/s10295-016-1802-2
 
[1]:  Chen, F., Li, W., Jiang, L., Pu, X., Yang, Y., & Zhang, G. (2016). Functional characterization of a geraniol synthase ‑ encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. https://doi.org/10.1007/s10295-016-1802-2
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[5]:xxxx
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2753003 parameters</partinfo>
 
<partinfo>BBa_K2753003 parameters</partinfo>
 
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Revision as of 09:07, 7 October 2018


pSB1C3-obGES cds

This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilica (sweet basil) and it’s codon-optimised for the use in E. coli. GES catalyses the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries[1], and it’s an intermediate to the biosynthesis of nepetalactol which is an active ingredient in catnip.

Characterization

In our study, we aim to achieve geraniol synthesis in E. coli, so at the beginning, we expressed a geraniol synthesis operon (https://parts.igem.org/Parts:BBa_K2753016) containing an Abies Grandis geranyl pyrophosphate synthase (https://parts.igem.org/Parts:BBa_K2753002) and an Ocimum basilicum geraniol synthase (https://parts.igem.org/Parts:BBa_K2753003) regulated by a pTac promoter in E. coli. Assuming that higher expression leads to higher yield, the operon was assembled on a high copy vector pUC20 whose copy number is approximately 500 per cell. However, when carrying out shake-flask fermentation with this strain, after induced by 25uM IPTG for 24 hours, no geraniol production was detected using gas chromatography. (see detailed methods in our notebook LINK) We infer that the failure to produce geraniol was due to the lack of substrates IPP and DMAPP. Thus we decided to co-express an MVA pathway in addition to the geraniol synthesis operon. The pathway can either be[5] added by cotransformation of a plasmid containing only the MVA pathway and the other plasmid with only GPPS and GES, or by constructing an all-in-one plasmid containing the MVA pathway, GPPS, and GES. We obtained a plasmid pMVA-only which was a gift from Taek Soon Lee, and have added GPPS and GES onto pMVA-only to acquire a pMVA-GPPS-GES.





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 30℃, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 449
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1135
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1161

Reference

[1]: Chen, F., Li, W., Jiang, L., Pu, X., Yang, Y., & Zhang, G. (2016). Functional characterization of a geraniol synthase ‑ encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. https://doi.org/10.1007/s10295-016-1802-2 [5]:xxxx