Difference between revisions of "Part:BBa K4880017"
 
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<partinfo>BBa_K4880017 short</partinfo>
 
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This composite part encodes for Theo-induced bPS and is composed of the basic parts theophylline inducible promoter and beta pinene synthase.  
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This composite part encodes for βPS and is composed of the basic parts theophylline inducible promoter and β-pinene synthase.  
  
 
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<partinfo>BBa_K4880017 parameters</partinfo>
 
<partinfo>BBa_K4880017 parameters</partinfo>
 
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===Assembly===
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===Plasmid construction===
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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.
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/betaps-plasmid.png" width = "50%"><br></html></center>
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<center>Figure 1: pPMQAK1-Ptrc-theo-βPS plasmid diagram</center>
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===Parts===
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===Theophylline inducible promoter===
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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.
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===β-pinene synthase===
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β-pinene synthase converts geranyl pyrophosphate to (-)-β-pinene and is isolated from Sitka spruce
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===Results===
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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. 
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/betaps-ecoli-gel.jpg" width = "40%"><br></html></center>
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<center>Figure 2: βPS colony PCR gel electrophoresis results (E. coli DH5α)</center>
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To further confirm the constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results. 
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/betaps-sequencing.png" width = "75%"><br></html></center>
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<center>Figure 3: sequencing results of pPMQAK1-Ptrc-theo-βPS</center> 
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After transforming pPMQAK1-Ptrc-theo-βPS into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results.
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/betaps-6803-gel.jpg" width = "40%"><br></html></center>
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<center>Figure 4: βPS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)</center> 
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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.
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/betaps-gcresults.png" width = "60%"><br></html></center>
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<center>Figure 5: βPS gas chromatography results</center>
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===References===
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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.

Latest revision as of 09:00, 12 October 2023


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

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.