Part:BBa_K274002:Experience

[http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia] have analysed products of this part along with products of Part:BBa_K274004 by thinlayer liquid cromatography and mass spectrometry (for a detailed description of the procedures see the 2010 iGEM team Slovenia [http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS/protocols/bm#TLC wiki]). Unfortunately we had problems with transforming Part:BBa_K274002, so we constructed our own vio operon from Part:BBa_K274003, Part:BBa_B0034 and a vioC gene, obtained by PCR from Part:BBa_K274004. Transcription of the construct therefore resulted in same proteins as Part:BBa_K274002.

[[Image:Tlc_(tinal).jpg |left|thumb|500px| Figure: TLC separation of culture extracts on a silica gel plate under white light and UV light.]

Results of our experiments show that both tested constructs result in formation of three main compounds, namely violacein, deoxyviolacein and deoxychromoviridans. Part:BBa_K274002 produces a large amount of deoxyviolacein, and smaller amounts of violacein and deoxychromoviridans, while Part:BBa_K274004 results in production of deoxychromoviridans and some deoxyviolacein rather than violacein.

Judging by the violacein biosynthesis pathway scheme uploaded on the main page, the results imply that spontaneus transformations of intermediates in the violacein biosynthetic pathway occure frequently, which reduces the biosynthetic flow of violacein and results in a lower yield of the latter. Since violacein is a potentially very applicable compound in terms of medicine and other applications (for instance natural purple dyes), it is interesting for industrial production, which could be problematic because of the unwanted side products like deoxychromoviridans. That is why [http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia] have decided to improve the violacein biosynthetic pathway, in attempts of which we used new constructs derived from Part:BBa_K274002 – we constructed chimeric vio enzymes, fused with zinc finger proteins (Part:BBa_K323132 and Part:BBa_K323135).

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