Part:BBa_K323135
VioA and VioB enzymes fused with zinc fingers under pBAD promoter
This part contains two of the enzymes involved in the violacein biosynthetic pathway, fused to zinc finger proteins. VioA enzyme is linked to the Blues zinc finger (Part:BBa_K323085) and VioB enzyme to the Zif268 zinc finger (Part:BBa_K323134). When translated into protein, two functional chimeric proteins, which bind to a specific DNA sequence (target sequence for binding of zinc fingers; check our DNA program for biosynthesis) 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_K323132, 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 and obtained 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 enzymatic 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, but still obtained more than 6-fold increase of violacein production with DNA program compared to no program. 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
Illegal NheI site found at 2568 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal BamHI site found at 6132 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 5657
Illegal AgeI site found at 979
Illegal AgeI site found at 5853 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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