Difference between revisions of "Part:BBa K3038002:Design"

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RFC10  standard
 
RFC10  standard
  
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In order to produce the molecule of interest 2-nonanone, we worked with the Lawrence Berkeley National Laboratory, USA which is working on biofuels and modified E. coli strain and obtain a production of 2-nonanone. This production is possible using free fatty acids as substrate.  
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In order to produce the molecule of interest 2-nonanone, we worked with the Lawrence Berkeley National Laboratory, USA which is working on biofuels and modified <i>E. coli</i> strain and obtain a production of 2-nonanone. This production is possible using free fatty acids as substrate.  
  
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Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in E. coli.  
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Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in <i>E. coli</i>.  
  
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Thanks to Geneious software we have designed a gene with a promoter, and a tag. This part doesn’t have a terminator because its produced to create a composite part with other gene involved in 2-nonanone synthesis. The promoter will therefore be associated with the design of the last gene of the composite part. The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university.  
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Thanks to Geneious software we have designed a gene with a promoter, and a tag. This part doesn’t have a terminator because its produced to create a composite part with other gene involved in 2-nonanone synthesis. The promoter will therefore be associated with the design of the last gene of the composite part (Mlut_11700: BBa_K3038004). The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university.  
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This part is already exciting with number. But we decided to improve it by adding a 6-his tag. This allows to purify and detect the protein in the host strain by using Ni-NTA columns.  
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This part is already exciting with number BBa_K1472601. But we decided to improve it by adding a 6-his tag. This allows to purify and detect the protein in the host strain by using Ni-NTA columns.  
  
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https://static.igem.org/mediawiki/parts/d/de/T--Poitiers--TesA_design-tab3.jpg
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<center>https://static.igem.org/mediawiki/parts/d/de/T--Poitiers--TesA_design-tab3.jpg<br>
  
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Following the design of the synthetic gene, It is amplified by PCR thanks to the design of primers upstream and downstream of the sequence. After amplification of the synthetic gene, sample is purified, the amplicons are digested with restriction enzymes EcoRI and PstI. Similarly for the cloning vector pSB1A3 according to the protocol described above. The insert (TesA) is then ligated into the plasmid.
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<strong>Design of the ADR-C-term gene with the Geneious Software.<br>
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The design show restriction enzymes like EcoRI, XbaI, SpeI and PstI. The gene includes pBAD promoter and its terminater. Finally 6-His tag are present in C-term.
  
 
===Source===
 
===Source===

Revision as of 15:03, 21 October 2019


Thioestherase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

RFC10 standard

In order to produce the molecule of interest 2-nonanone, we worked with the Lawrence Berkeley National Laboratory, USA which is working on biofuels and modified E. coli strain and obtain a production of 2-nonanone. This production is possible using free fatty acids as substrate.

Here we present the cloning of thioesterase I (TesA), an enzyme involved in the synthesis of free fatty acids in E. coli.

Thanks to Geneious software we have designed a gene with a promoter, and a tag. This part doesn’t have a terminator because its produced to create a composite part with other gene involved in 2-nonanone synthesis. The promoter will therefore be associated with the design of the last gene of the composite part (Mlut_11700: BBa_K3038004). The promoter is inducible to arabinose. This allows a controlled expression of the synthetic gene to avoid any effect of toxicity. In addition, arabinose is an inexpensive inducer and very present in the laboratories of our university. This part is already exciting with number BBa_K1472601. But we decided to improve it by adding a 6-his tag. This allows to purify and detect the protein in the host strain by using Ni-NTA columns.

T--Poitiers--TesA_design-tab3.jpg

Design of the ADR-C-term gene with the Geneious Software.
The design show restriction enzymes like EcoRI, XbaI, SpeI and PstI. The gene includes pBAD promoter and its terminater. Finally 6-His tag are present in C-term.

Source

DNA synthesis

RFC10 Standard

GC content optimized

References

Engineering of Bacterial Methyl Ketone Synthesis for Biofuels. Ee-Been Goh,a,c Edward E. K. Baidoo,a,c Jay D. Keasling,a,c,d and Harry R. Beller. Appl Environ Microbiol. 2012 Jan; 78(1): 70–80. doi: 10.1128/AEM.06785-11. PMCID: PMC3255637. PMID: 22038610