Part:BBa_K323132
VioC, VioD and VioE enzymes fused with zinc fingers under pBAD promoter
This part contains three of the enzymes of the violacein biosynthetic pathway, fused to zinc finger proteins. VioC enzyme is linked to the Gli1 zinc finger (Part:BBa_K323086), vioD to HivC zinc finger (Part:BBa_K323087) and VioE enzyme to the PBSII zinc finger (Part:BBa_K323131). When translated to protein, three functional chimeric proteins which bind to a specific DNA sequence are produced.
Genes for violacein biosynthesis are arranged in an operon consisting of vioA, vioB, vioC, vioD and vioE. VioA generates an IPA imine from L-tryptophan and VioB converts the IPA imine into a dimer. VioE then acts by transforming the dimer into protodexyviolaceinic acid (PVA), which can be spontaneously converted into a green pigment called deoxychromoviridans. VioD and VioC hydroxylate PVA to form violacein.
The 2010 iGEM team Slovenia improved the rather poor yield of violacein production in E.coli and reduced the formation of the unwanted side product (deoxychromoviridans) by introducing the DNA-guided biosynthetic pathway based on chimeric enzymes bound in correct order to the DNA program.
In combination with Part:BBa_K323135, we have bound all of the enzymes, necessary for synthesis of violacein, on a DNA program (Part:BBa_K323066). This enabled a faster and increased synthesis of the desired product, as the enzymes were fixed close together and the intermediates were immediately available to the next enzyme. The latter fact also resulted in a decreased formation of deoxychromoviridans, which most likely also contributed to the overall increased production of violacein. We have also designed a scrambled DNA program (Part:BBa_K323153) to demonstrate the importance of enzyme order for the efficiency of violacein production.
We have conducted our experiments several times with similar results. Although the theoretical increase of biosynthetic reaction is significantly higher for the chain of five reactions, the final yield depends on many different factors, such as rate limiting steps, like availability of the substrate (e.g. low solubility), cofactors etc. We did not perform any optimizations with respect to strain, growth media, temperature etc. Results of our experiments are diplayed on the Experience page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1204
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3078
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 3177 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI.rc site found at 4320
Illegal SapI.rc site found at 4395
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