Difference between revisions of "Part:BBa K2753015"

 
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<partinfo>BBa_K2753015 short</partinfo>
 
<partinfo>BBa_K2753015 short</partinfo>
  
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This composite part is designed for users to choose the strength of expression of geranyl diphosphate(GPP) and geraniol.  
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This part consists of a ribozyme (BBa_K1679038), two RBSs, an <em>Abies Grandis</em> geranyl pyrophosphate synthase coding sequence (BBa_K2753002) and an <em>Ocimum basilicum<em> geraniol synthase (BBa_2753003)It metabolizes IPP and DMAPP which are five-carbon blocks produced by either MVA pathway or MEP pathway or in some case both MVA and MEP pathway. IPP and DMAPP are converted to geraniol, a monoterpenoid with a rose-like odour and is a prosperous antimicrobial reagent and gasoline alternative. With addition of more downstream enzymes, geraniol can be changed to a large range of monoterpenes like linalool, actinide and nepetalactol.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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===Characterization===
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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) </p><br>
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<p> 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 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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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/parts/f/f7/T--GreatBay_China--pMVA-GPPS-GES.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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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/parts/7/7c/T--GreatBay_China--pMVA-only.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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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/parts/5/58/T--GreatBay_China--_MVA_only_and_MVAGG_Geraniol_yield.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> Under the same fermentation condition, pMVA-GPPS-GES gave a geraniol titer of 13.3mg/L while the negative control pMVA-only as well produced geraniol titer of about 5.7mg/L. Then we are assured that a stable supply of precursors IPP and DMAPP are crucial for geraniol production. But still, there is room for higher geraniol yield. Therefore, to gain maximum flux to geraniol, we tuned the expression level of GPPS and GES using three promoters:</p>
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<ul>
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<li>pTALE sp1 (https://parts.igem.org/Part:BBa_K2753030), copy number-independent promoter of medium strength</li>
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<li>pTALE sp2 (https://parts.igem.org/Part:BBa_K2753018), 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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<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>Having transferred the constructs above along with pMVA-only into strain E. coli DH5α, the same shake-flask fermentation experiment was conducted. The result shows detectable geraniol production by each strain.</p>
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 +
<html>
 +
<center>
 +
<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/parts/8/88/T--GreatBay_China--geraniol_p_table.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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<html>
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<center>
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<Figure>
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<img width="70%" src="https://static.igem.org/mediawiki/parts/7/7e/T--GreatBay_China--geraniol_d.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> With pTac promoters, geraniol yield increased with the copy number of the vector, showing positively correlated relation. However, when TALE stabilized promoter (TALEsp) was used, higher the copy number of the vector, lower the production of geraniol, being the opposite of pTac. And the stronger TALEsp promoter, TALE1sp1 gave generally reduced yield compared to its weaker counterpart TALE2sp1. Interestingly, the graphs of the two TALEsp promoters appeared to be seemingly parallel to each other. </p>
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 +
<p> We surmised that the yield of geraniol was affected by two factors: the expression level of enzymes and the cellular burden. As for enzyme expression, there exists an optimal gene expression level that produces just enough enzyme to metabolize all the substrate. The yield would be the greatest at this level. But if lower than this level, production would increase with enzyme expression since there is a surplus of substrates. And if the enzyme expression is higher, the more enzymes now becomes a cellular burden as it no longer contributes to more product.</p>
 +
 
 +
<p> In the case of pTac, it fits into the scenario when the gene expression is lower than the optimum: higher copy produced more enzymes to catalyze geraniol synthesis reaction. As for TALE stabilized promoters, the expression level of enzymes would remain unchanged regardless of copy number due to the stabilization nature of pTALE (LINK more about pTALE). So in low copy vector where the cellular burden isn’t significant, the product yield is only related to the strength of the promoter, explaining why pTALE sp2 has better performance than pTALE sp1 as it’s closer to the optimum. But when the copy number increases, the expression level is unaffected while the cellular burden rises sharply because much more TALE would be made to stabilize expression, leaving less energy available for geraniol synthesis. And it in turns answers why a negative trend of production is shown when regulated by pTALE.</p>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2753015 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2753002 SequenceAndFeatures</partinfo>
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===Referece===
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[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
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[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
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2753015 parameters</partinfo>
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<partinfo>BBa_K2753002 parameters</partinfo>
 
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Revision as of 07:12, 9 October 2018


pSB1C3-RiboJ-rbs-GPPS-rbs-ObGES

This part consists of a ribozyme (BBa_K1679038), two RBSs, an Abies Grandis geranyl pyrophosphate synthase coding sequence (BBa_K2753002) and an Ocimum basilicum geraniol synthase (BBa_2753003)It metabolizes IPP and DMAPP which are five-carbon blocks produced by either MVA pathway or MEP pathway or in some case both MVA and MEP pathway. IPP and DMAPP are converted to geraniol, a monoterpenoid with a rose-like odour and is a prosperous antimicrobial reagent and gasoline alternative. With addition of more downstream enzymes, geraniol can be changed to a large range of monoterpenes like linalool, actinide and nepetalactol.


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 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.





Under the same fermentation condition, pMVA-GPPS-GES gave a geraniol titer of 13.3mg/L while the negative control pMVA-only as well produced geraniol titer of about 5.7mg/L. Then we are assured that a stable supply of precursors IPP and DMAPP are crucial for geraniol production. But still, there is room for higher geraniol yield. Therefore, to gain maximum flux to geraniol, we tuned the expression level of GPPS and GES using three promoters:


And three vectors of unique copy number:


  • pUC20: ~500
  • pR6K: ~15
  • pSC101: ~1



Having transferred the constructs above along with pMVA-only into strain E. coli DH5α, the same shake-flask fermentation experiment was conducted. The result shows detectable geraniol production by each strain.



With pTac promoters, geraniol yield increased with the copy number of the vector, showing positively correlated relation. However, when TALE stabilized promoter (TALEsp) was used, higher the copy number of the vector, lower the production of geraniol, being the opposite of pTac. And the stronger TALEsp promoter, TALE1sp1 gave generally reduced yield compared to its weaker counterpart TALE2sp1. Interestingly, the graphs of the two TALEsp promoters appeared to be seemingly parallel to each other.

We surmised that the yield of geraniol was affected by two factors: the expression level of enzymes and the cellular burden. As for enzyme expression, there exists an optimal gene expression level that produces just enough enzyme to metabolize all the substrate. The yield would be the greatest at this level. But if lower than this level, production would increase with enzyme expression since there is a surplus of substrates. And if the enzyme expression is higher, the more enzymes now becomes a cellular burden as it no longer contributes to more product.

In the case of pTac, it fits into the scenario when the gene expression is lower than the optimum: higher copy produced more enzymes to catalyze geraniol synthesis reaction. As for TALE stabilized promoters, the expression level of enzymes would remain unchanged regardless of copy number due to the stabilization nature of pTALE (LINK more about pTALE). So in low copy vector where the cellular burden isn’t significant, the product yield is only related to the strength of the promoter, explaining why pTALE sp2 has better performance than pTALE sp1 as it’s closer to the optimum. But when the copy number increases, the expression level is unaffected while the cellular burden rises sharply because much more TALE would be made to stabilize expression, leaving less energy available for geraniol synthesis. And it in turns answers why a negative trend of production is shown when regulated by pTALE.

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]

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