Composite

Part:BBa_K4880015

Designed by: Xinran Xia   Group: iGEM23_BASIS-China   (2023-09-08)


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.

References

Blanc-Garin V, Chenebault C, Diaz-Santos E, Vincent M, Sassi JF, Cassier-Chauvat C, Chauvat F. Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes. Biotechnol Biofuels Bioprod. 2022 Oct 14;15(1):110.

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