Difference between revisions of "Part:BBa K4880004"
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===Results=== | ===Results=== | ||
− | After transforming pPMQAK1-Ptrc-theo-PaFS 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-PaFS 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. |
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-ecoli-gel.png" width = "30%"><br></html></center> | <center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-ecoli-gel.png" width = "30%"><br></html></center> | ||
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<center>Figure 3: sequencing results of pPMQAK1-Ptrc-theo-PaFS</center> | <center>Figure 3: sequencing results of pPMQAK1-Ptrc-theo-PaFS</center> | ||
− | To detect whether the farnesene synthase genes are expressed, we assembled the | + | To detect whether the farnesene synthase genes are expressed, we assembled the farnesene 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-PaFS into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results. | + | <center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-proteincheck.png" width = "15%"><br></html></center> |
+ | <center>Figure 4: SDS-PAGE protein check gel electrophoresis result of PaFS</center> | ||
+ | |||
+ | After transforming pPMQAK1-Ptrc-theo-PaFS into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results. | ||
<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-6803-gel.png" width = "30%"><br></html></center> | <center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-6803-gel.png" width = "30%"><br></html></center> | ||
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To test whether farnesene is produced, we performed gas chromatography with the help of our advisors. The results below show that we successfully produced farnesene in Synechocystis sp. PCC 6803. | To test whether farnesene is produced, we performed gas chromatography with the help of our advisors. The results below show that we successfully produced farnesene in Synechocystis sp. PCC 6803. | ||
+ | |||
+ | <center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/pafs-gcresults.png" width = "60%"><br></html></center> | ||
+ | <center>Figure 6: PaFS gas chromatography results</center> | ||
+ | |||
+ | ===References=== | ||
+ | |||
+ | Blanc-Garin V, Chenebault C, Diaz-Santos E, Vincent M, Sassi JF, Cassier-Chauvat C, Chauvat F. Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes. Biotechnol Biofuels Bioprod. 2022 Oct 14;15(1):110. |
Latest revision as of 09:01, 12 October 2023
Ptrc-theo-PaFS
This composite part encodes for PaFS and is composed of the basic parts theophylline inducible promoter and farnesene synthase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1071
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1220
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 55
- 1000COMPATIBLE WITH RFC[1000]
Assembly
Plasmid construction
Through homologous recombination, we integrated the farnesene 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.
Farnesene synthase
Farnesene synthase converts farnesyl pyrophosphate to α-farnesene and is isolated from Picea abies.
Results
After transforming pPMQAK1-Ptrc-theo-PaFS 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.
To detect whether the farnesene synthase genes are expressed, we assembled the farnesene 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-PaFS into Synechocystis sp. PCC 6803, we performed colony PCR. Below are the results.
To test whether farnesene is produced, we performed gas chromatography with the help of our advisors. The results below show that we successfully produced farnesene in Synechocystis sp. PCC 6803.
References
Blanc-Garin V, Chenebault C, Diaz-Santos E, Vincent M, Sassi JF, Cassier-Chauvat C, Chauvat F. Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes. Biotechnol Biofuels Bioprod. 2022 Oct 14;15(1):110.