Difference between revisions of "Part:BBa K3038002"

 
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TesA : GenBank: EG11542
 
TesA : GenBank: EG11542
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Cloning of Acyl ACP thioesterase gene for 2-nonanone production
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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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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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===Usage and Biology===
 
===Usage and Biology===
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===Design===
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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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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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https://parts.igem.org/File:T--Poitiers--TesA_design-tab3.jpg
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===PCR Amplification===
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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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Revision as of 10:03, 16 October 2019

TesA : GenBank: EG11542

Cloning of Acyl ACP thioesterase gene for 2-nonanone production

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.



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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]