Difference between revisions of "Part:BBa K4880016"

(→‎Plasmid construction)
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After transforming pPMQAK1-Ptrc-theo-αPS 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.   
 
After transforming pPMQAK1-Ptrc-theo-αPS 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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<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/alphaps-ecoli-gel.png" width = "50%"><br></html>
  
 
To further confirm the constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results.   
 
To further confirm the constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results.   

Revision as of 16:11, 8 October 2023


Ptrc-theo-αPS

This composite part encodes for αPS and is composed of the basic parts theophylline inducible promoter and α-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 1361
    Illegal XhoI site found at 1441
  • 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 α-pinene synthase gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.

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.

α-pinene synthase

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

Results

After transforming pPMQAK1-Ptrc-theo-αPS 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.


To further confirm the constructed plasmids are correct, we sent them to be sequenced. Below are the sequencing results.

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

To test whether α-pinene is produced, we plan on performing gas chromatography with the help of our advisors.