Part:BBa_K4880005

SaSS

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1023
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1023
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1023
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1023
  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
  

Plasmid construction

Through homologous recombination, we integrated the santalene 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-SaSS plasmid diagram

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 santalene and is isolated from Sitka spruce Santalum Album.

Results

After transforming pPMQAK1-Ptrc-theo-SaSS 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: SaSS 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-SaSS

To detect whether the santalene synthase genes are expressed, we assembled the santalene synthase gene with E.coli expression plasmid, pET28a. We then transformed this plasmid into E.coli BL21 (DE3) and expressed this protein at 0.5 mM IPTG at 37◦C. The figure below shows the SDS-PAGE results.

Figure 4: SDS-PAGE protein check gel electrophoresis result of SaSS

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

Figure 5: SaSS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)

To test whether santalene 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.