Difference between revisions of "Part:BBa K4880006"
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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.  
 
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.  
  
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<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-plasmid.png" width = "50%"><br></html>
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-plasmid.png" width = "50%"><br></html></center>
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<center>Figure 1: pPMQAK1-Ptrc-theo-AgBS plasmid diagram</center>
  
 
===Parts===
 
===Parts===
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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.   
 
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.   
  
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<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-ecoli-gel.jpg" width = "40%"><br></html>
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-ecoli-gel.jpg" width = "40%"><br></html></center>
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<center>Figure 2: AgBS colony PCR gel electrophoresis results (E. coli DH5α)</center>
  
 
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.   
  
−
<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-sequencing.png" width = "75%"><br></html>   
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-sequencing.png" width = "75%"><br></html></center>
 +
<center>Figure 3: sequencing results of pPMQAK1-Ptrc-theo-AgBS (E. coli DH5α)</center>   
  
 
After transforming pPMQAK1-Ptrc-theo-AgB into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results.  
 
After transforming pPMQAK1-Ptrc-theo-AgB into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results.  
  
−
<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-6803-gel.png" width = "40%"><br></html>  
+
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/agbs-6803-gel.png" width = "40%"><br></html></center>
 +
<center>Figure 4: AgBS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)</center>
  
 
To test whether bisabolene is produced, we plan on performing gas chromatography with the help of our advisors.
 
To test whether bisabolene is produced, we plan on performing gas chromatography with the help of our advisors.

Revision as of 08:18, 10 October 2023


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


Assembly

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.


Figure 1: pPMQAK1-Ptrc-theo-AgBS 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 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.


Figure 2: AgBS 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-AgBS (E. coli DH5α)

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


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

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