Part:BBa_K4880007

Ptrc-theo-PtPS

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1322
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 55
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1989

Assembly

Plasmid construction

Through homologous recombination, we integrated the pinene 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-PtPS 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.

Pinene synthase

Pinene synthase converts geranyl pyrophosphate to (+)-alpha-pinene and is isolated from Pinus taeda.

Results

After transforming pPMQAK1-Ptrc-theo-PtPS 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: PtPS 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-PtPS (E. coli DH5α)

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

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

To test whether pinene 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.