Difference between revisions of "Part:BBa K1065106"
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===Characterization=== | ===Characterization=== | ||
Revision as of 12:28, 4 October 2013
Wintergreen device with SAM: Plac + PCHBA + araCpBAD+ BSMT1 + Plac + SAM synthetase
This is the complete wintergreen odor device from iGEM UniTN-Trento 2013 which encodes for four enzymes: PchB, PchA, BSMT1 and SAM. The device produces methyl salicylate (a fruit ripening inhibitor) [1]and was used for the development of the project B. Fruity by UniTN Trento 2013. This part, containing SAM enzyme BBa_K1065105 [5], is an improvement of part BBa_K1065102.
Usage and Biology
The chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria have, undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA [2]. Salicylate is then obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism Pseudomonas aeruginosa.[3] In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 682
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 223
Characterization
MeSA detection
NEB10β cells transformed with Bba_K1065106, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 0.9 ppm for Bba_K1065106 (in the presence of salicylic acid). Non induced cells did not produce any MeSA.
Figure 2 Quantification of MeSA by gas chromatography. a)NEB10β cells transformed with Bba_K1065106 and induced with 5mM arabinose and supplemented with salycilic acid, b) 15 ppm reference point.