Difference between revisions of "Part:BBa K4880005"
Line 23: Line 23:
 
Through homologous recombination, we integrated the santalene 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 santalene synthase gene into the broad host range replicative vector pPMQAK1 along with the theophylline inducible promoter. The following figure shows the recombinant plasmid.  
  
−
<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-plasmid.png" width = "50%"><br></html>
+
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-plasmid.png" width = "50%"><br></html></center>
 +
<center>Figure 1: pPMQAK1-Ptrc-theo-SaSS plasmid diagram</center>
  
 
===Parts===
 
===Parts===
Line 38: Line 39:
 
After transforming pPMQAK1-Ptrc-theo-SaSS 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-SaSS 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.   
  
−
<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-ecoli-gel.jpg" width = "30%"><br></html>
+
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-ecoli-gel.jpg" width = "30%"><br></html></center>
 +
<center>Figure 2: SaSS 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/sass-sequencing.png" width = "75%"><br></html>
+
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-sequencing.png" width = "75%"><br></html></center>
 +
<center>Figure 3: sequencing results of pPMQAK1-Ptrc-theo-SaSS</center>
  
 
To detect whether the santalene synthase genes are expressed, we assembled the santalene synthase gene with E.coli expression plasmid, pET28a. We then transformed this plasmid into E.coli BL21 (DE3) and expressed this protein at 0.5 mM IPTG at 37◦C. The figure below shows the SDS-PAGE results.  
 
To detect whether the santalene synthase genes are expressed, we assembled the santalene synthase gene with E.coli expression plasmid, pET28a. We then transformed this plasmid into E.coli BL21 (DE3) and expressed this protein at 0.5 mM IPTG at 37◦C. The figure below shows the SDS-PAGE results.  
Line 48: Line 51:
 
After transforming pPMQAK1-Ptrc-theo-SaSS into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results.   
 
After transforming pPMQAK1-Ptrc-theo-SaSS into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results.   
  
−
<html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-6803-gel.png" width = "30%"><br></html>  
+
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/sass-6803-gel.png" width = "30%"><br></html></center>
 +
<center>Figure 5: SaSS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)</center>
  
 
To test whether santalene is produced, we plan on performing gas chromatography with the help of our advisors.
 
To test whether santalene is produced, we plan on performing gas chromatography with the help of our advisors.

Revision as of 08:14, 10 October 2023


SaSS

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

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1023
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1023
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1023
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1023
    Illegal AgeI site found at 55
    Illegal AgeI site found at 984
    Illegal AgeI site found at 1623
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1418


Assembly

Plasmid construction

Through homologous recombination, we integrated the santalene 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-SaSS 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.

Santalene synthase

Santalene synthase converts farnesyl pyrophosphate to santalene and is isolated from Sitka spruce Santalum Album.

Results

After transforming pPMQAK1-Ptrc-theo-SaSS 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: SaSS 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-SaSS

To detect whether the santalene synthase genes are expressed, we assembled the santalene synthase gene with E.coli expression plasmid, pET28a. We then transformed this plasmid into E.coli BL21 (DE3) and expressed this protein at 0.5 mM IPTG at 37◦C. The figure below shows the SDS-PAGE results.

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


Figure 5: SaSS colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)

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