4-DG Pathway (E.coli Optimised)
The following part describes the constitutive expression of the first two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobricks BBa_K3634000 (DHQS) and BBa_K3634001 (O-MT) are responsible for converting sedoheptulose 7-phosphate to 4-deoxygadusol. Once produced, 4-deoxygadusol will then be converted by BBa_K3634005 (ATPG and NRPS composite, present on 'plasmid A') to the final product of the pathway, shinorine. The shinorine production pathway is separated in this way as a biosafety mechanism so that UV resistance is not conferred in a bacteria which has lost or gained plasmid A/B alone. As NRPS is determined to be 'rate-limiting' with respect to the pathway, plasmid A will ideally be placed at a higher copy number to plasmid B to ensure 1:1 stoichiometry of reactants.
For our bacteria to provide maximum expression of the UV absorbing compound shinorine, the strong constitutive promoter BBa_J23119 was selected from the Anderson promoter catalogue alongside RBS BBa_B0034 inserted between each ORF. These parts are a preliminary choice based off previous experimental expression data (Berkeley iGEM, 2006). The commonly used double terminator BBa_B0015 will ensure reliable release of the new mRNA strand.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC