Part:BBa_K274002:Experience

[http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia] has analysed products of this part along with products of Part:BBa_K274004 by thinlayer liquid chromatography 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 transformation of Part:BBa_K274002 in E.coli, so we constructed our own vio operon from Part:BBa_K274003, Part:BBa_B0034 and a vioC gene, amplified by PCR from Part:BBa_K274004. Transcription of the construct therefore resulted in same proteins as Part:BBa_K274002.

Figure: TLC separation of culture extracts on a silica gel plate under white light and UV light.

Results of our experiments show that both of the tested constructs lead to synthesis of three main compounds, namely violacein, deoxyviolacein and deoxychromoviridans (this was confirmed with standards and MS). 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 pathways scheme uploaded on the main page, the results imply that spontaneous transformations of intermediates in the violacein biosynthetic pathway occur frequently, therefore reducing the biosynthetic flow towards violacein and resulting 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 that could be problematic because of the unwanted side products like deoxychromoviridans. That is why [http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia] has decided to improve the violacein biosynthetic pathway. Therefore we designed new constructs derived from Part:BBa_K274002. We designed chimeric vio enzymes, fused with zinc finger proteins (Part:BBa_K323132 and Part:BBa_K323135). We showed higher yields of violacein production where biosynthetic enzymes are bound to zinc fingers and through them to DNA scaffold (called DNA program). DNA program was designed in such a way, that it arranges biosynthetic enzymes in a correct order, leading to close proximity which ensures higher yields of biosynthesis and decreases formation of side products. See our project on [http://2010.igem.org/Team:Slovenia 2010 iGEM team Slovenia].

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