Composite

Part:BBa_K4880017

Designed by: Xinran Xia   Group: iGEM23_BASIS-China   (2023-09-08)
Revision as of 07:50, 10 October 2023 by Vivixia (Talk | contribs)


Ptrc-theo-βPS

This composite part encodes for βPS and is composed of the basic parts theophylline inducible promoter and β-pinene 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
    COMPATIBLE WITH RFC[1000]


Assembly

Plasmid construction

Through homologous recombination, we integrated the β-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-βPS 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 β-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.

β-pinene synthase

β-pinene synthase converts geranyl pyrophosphate to (-)-β-pinene and is isolated from Sitka spruce

Results

After transforming Ptrc-theo-βPS 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: βPS 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-βPS

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


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

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


Figure 5: βPS gas chromatography results
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Parameters
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