Composite

Part:BBa_K4880004

Designed by: Xinran Xia   Group: iGEM23_BASIS-China   (2023-08-26)
Revision as of 12:44, 11 October 2023 by Vivixia (Talk | contribs) (→‎Results)


Ptrc-theo-PaFS

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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1071
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1220
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 55
  • 1000
    COMPATIBLE WITH RFC[1000]


Assembly

Plasmid construction

Through homologous recombination, we integrated the farnesene 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-PaFS 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.

Farnesene synthase

Farnesene synthase converts farnesyl pyrophosphate to α-farnesene and is isolated from Picea abies.

Results

After transforming pPMQAK1-Ptrc-theo-PaFS 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: PaFS 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-PaFS

To detect whether the farnesene synthase genes are expressed, we assembled the farnesene 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 PaFS

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


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

To test whether farnesene is produced, we performed gas chromatography with the help of our advisors. The results below show that we successfully produced farnesene in Synechocystis sp. PCC 6803.


Figure 6: PaFS gas chromatography results
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