ATPG and NRPS Composite (E.coli Optimised)
The following part describes the constitutive expression of the final two enzymes of the shinorine production pathway, optimised for use in E.coli. Within the composite part, biobrick BBa_K3634002 (ATPG) and BBa_K3634003 (NRPS) are responsible for converting 4-deoxygadusol (4-DG) to the final product shinorine. Once produced, this mycosporine-like amino acid can absorb UV radiation of wavelength ~333nm. The composite part described will be present on 'plasmid A', separate to the previous enzymes responsible for 4-deoxygadusol production which are maintained on 'plasmid B'. By this arrangement, biosafety of our gene circuit will be ensured such 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.
The enzyme ATPG was included on plasmid A with NRPS instead of on plasmid B with DHQS and O-MT as mycosporine glycine, an intermediate which also absorbs UV radiation, would have been produced. If a bacteria by chance was transformed with this three enzyme plasmid B variant, the organism would be UV resistant to a certain degree which therefore would confer a survival advantage in the environment.
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. These parts are a preliminary choice based off previous experimental expression data (Berkeley iGEM, 2006). The commonly used double terminator BBa_B0015 will also ensure reliable termination.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1561
Illegal NheI site found at 1584
- 21Illegal BglII site found at 2059
Illegal BamHI site found at 4112
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC