Difference between revisions of "Part:BBa K3038002"
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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. | 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. | ||
| + | The PCR product as well as the digestion products are deposited on 0.8 % agarose gel. In well 2, the TesA tagged with 6 his in C-ter amplified by PCR. The most intense band observed corresponds to the size expected for TesA around 900 pb. Another band, this time very weak, is visible below 400 pb. This band may be due to a specific pairing of the primers. | ||
| + | https://parts.igem.org/File:T--Poitiers--TesA_amplification-tab3.png | ||
Revision as of 10:05, 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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]