Part:BBa_K4880006

Ptrc-theo-AgBS

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1842
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1842
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1437
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1842
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1842
  Illegal AgeI site found at 55
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 225
  Illegal BsaI.rc site found at 666
  Illegal BsaI.rc site found at 1449
  

Plasmid construction

Through homologous recombination, we integrated the bisabolene synthase gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.

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.

Bisabolene synthase

Bisabolene synthase converts farnesyl pyrophosphate to E-α-bisabolene and is isolated from Abides grandis.

Results

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

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