Difference between revisions of "Part:BBa K2753003"
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<partinfo>BBa_K2753003 short</partinfo>
 
<partinfo>BBa_K2753003 short</partinfo>
  
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Geraniol synthase from Ocimum basilicum (sweet basil) converts geranyl pyrophosphate (GPP) to geraniol.
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This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilicum (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<sup>[1]</sup>, and it’s an intermediate to the biosynthesis of nepetalactol which is an active ingredient in catnip.
  
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<!-- Add more about the biology of this part here
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===Characterization===
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===Usage and Biology===
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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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<ul>
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<li>pTALE sp1 (https://parts.igem.org/Part:BBa_K2753000), copy number-independent promoter of medium strength</li>
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<li>pTALE sp2 (https://parts.igem.org/Part:BBa_K2753001), copy number-independent promoter of low strength</li>
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<li>pTac (https://parts.igem.org/Part:BBa_K180000), IPTG inducible promoter</li>
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</ul>
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<br>
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<p>And three vectors of unique copy number:</p><br>
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<ul>
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<li>pUC20: ~500</li>
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<li>pR6K: ~15</li>
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<li>pSC101: ~1<li>
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</ul>
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<p>In total, we obtained nine combinations of promoters and vectors which regulate the expression of GPPS with GES. </p>
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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/2018/a/a0/T--GreatBay_China--_3_promoters%2B3backbone.png">
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</figure>
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</center>
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</html>
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<br/>
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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>
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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/2018/3/38/T--GreatBay_China--_Geraniol_production.jpg">
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</figure>
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</center>
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</html>
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<br/>
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<p>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.</p>
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2753003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2753003 SequenceAndFeatures</partinfo>
  
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===Reference===
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[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
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 10:48, 1 October 2018


pSB1C3-obGES cds

This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilicum (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, 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:



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 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.


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