Difference between revisions of "Part:BBa K323132"

Revision as of 21:47, 27 October 2010

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.

Figure: The DNA-guided violacein biosynthetic pathway..

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 strains, growth media, temperature etc. Results of our experiments are diplayed on the Experience page.

Sequence and Features