Part:BBa_K4880018

Ptrc-theo-MsLIMS

This composite part encodes for MsLIMS and is composed of the basic parts theophylline inducible promoter and limonene 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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1960
  

Plasmid construction

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

(plasmid diagram)

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.

MsLIMS

Limone synthase converts geranyl pyrophosphate to limonene and is isolated from Mentha spicata.

Results

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

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