Difference between revisions of "Part:BBa K4880015"

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Through homologous recombination, we integrated the santalene and santalol synthases gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.  
 
Through homologous recombination, we integrated the santalene and santalol synthases gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.  
  
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<center>(plasmid diagram)</center>
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/cyp-plasmid.png" width = "50%"><br></html></center>
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<center>Figure 1: pPMQAK1-Ptrc-theo-SaSS-CYP plasmid diagram</center>
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===Parts===
 
===Parts===
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===Results===
 
===Results===
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After transforming pPMQAK1-Ptrc-theo-SaSS-CYP into E. coli DH5α, we performed colony PCR on the monocultures and selected the successfully transformed ones for amplification and extraction to later transform it into Synechocystis sp. PCC 6803. The figure below shows the colony PCR results. 
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To confirm that our constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results. 
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/cyp-sequencing.png" width = "75%"><br></html></center>
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<center>Figure 2: sequencing results of pPMQAK1-Ptrc-theo-SaSS-CYP</center> 
  
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To further confirm the constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results.  
+
After transforming pPMQAK1-Ptrc-theo-SaSS-CYP into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results.
  
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After transforming pPMQAK1-Ptrc-theo-SaSS-CYP into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results. 
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/cyp-6803-gel.png" width = "30%"><br></html></center> 
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<center>Figure 3: SaSS-CYP colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)</center>
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To test whether santalol is produced, we plan on performing gas chromatography with the help of our advisors.
 
To test whether santalol is produced, we plan on performing gas chromatography with the help of our advisors.

Revision as of 13:36, 11 October 2023


Ptrc-theo-SaSS-CYP

This composite part encodes for the santalene and santalol synthases and is composed of the basic parts theophylline inducible promoter, santalene synthase and santalol synthase.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3105
    Illegal PstI site found at 1023
    Illegal PstI site found at 2853
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3105
    Illegal PstI site found at 1023
    Illegal PstI site found at 2853
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3105
    Illegal XhoI site found at 2441
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3105
    Illegal PstI site found at 1023
    Illegal PstI site found at 2853
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3105
    Illegal PstI site found at 1023
    Illegal PstI site found at 2853
    Illegal NgoMIV site found at 1942
    Illegal NgoMIV site found at 2254
    Illegal AgeI site found at 55
    Illegal AgeI site found at 984
    Illegal AgeI site found at 1623
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1418
    Illegal BsaI site found at 2553



Assembly

Plasmid construction

Through homologous recombination, we integrated the santalene and santalol synthases gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.


Figure 1: pPMQAK1-Ptrc-theo-SaSS-CYP plasmid diagram


Parts

Theophylline inducible promoter

We decided to use an induction system composed of Ptrc promoter and theophylline dependent riboswitch theo E* to control the expression of the α-pinene synthase. The Ptrc promoter is a hybrid of lac and trp, making it stronger than the lac promoter. Transcription is regulated by IPTG and translation initiates only when there is theophylline present. This double regulation strictly regulates gene expression.

Santalene synthase

Santalene synthase converts farnesyl pyrophosphate to limonene and is isolated from Santalum Album.

Santalol synthase

Santalol sythase converts santalene to santalol. It is a hemoprotein of the Cytochrome P450 family derived form Santalum album.

Results

To confirm that our constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results.


Figure 2: sequencing results of pPMQAK1-Ptrc-theo-SaSS-CYP

After transforming pPMQAK1-Ptrc-theo-SaSS-CYP into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results.


Figure 3: SaSS-CYP colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)


To test whether santalol is produced, we plan on performing gas chromatography with the help of our advisors.