Composite

Part:BBa_K4880018

Designed by: Xinran Xia   Group: iGEM23_BASIS-China   (2023-09-08)
Revision as of 09:00, 12 October 2023 by Aeolus (Talk | contribs)

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Ptrc-theo-MsLIMS

This composite part encodes for MsLIMS and is composed of the basic parts theophylline inducible promoter and limonene synthase.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 55
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1960



Assembly

Plasmid construction

Through homologous recombination, we integrated the limonene synthase 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-MsLIMS 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.

MsLIMS

Limone synthase converts geranyl pyrophosphate to limonene and is isolated from Mentha spicata.

Results

After transforming pPMQAK1-Ptrc-theo-MsLIMS 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.


Figure 2: MsLIMS colony PCR gel electrophoresis results (E. coli DH5α)

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


Figure 3: sequencing results of pPMQAK1-Ptrc-theo-MsLIMS

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


Figure 4: MsLIMS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)

To test whether limonene 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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