Difference between revisions of "Part:BBa K814004"
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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. | 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. | ||
| − | == ATPG and NRPS Composite (E.coli | + | == ATPG and NRPS Composite (Optimised for E.coli) == |
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 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. | ||
Latest revision as of 14:36, 9 August 2020
mycosporine-glycine biosynthetic pathway
Our research has focused on two novel biosynthetic pathways found in two distinct algal species. A pathway ending in the production of two UV-protective compounds, shinorine and mycosporine-glycine, was cloned from Anabaena varibalis. DHQS catalyzes the first step in the pathway, converting sedoheptulose-7-phosphate into dehydroquinate. Dehydroquinate-O-methyltransferase (O-MT) then generates 4-deoxygadusol, which is converted to mycosprine-glycine by ATP-grasp (ATPG).
This composite part contains protein generators for DHQS, O-MT and ATPG. E. coli transformed with this composite part are able to synthesize mycosporine-glycine, as detected by HPLC analysis of the media. 4-deoxygadusol is also detected in HPLC analysis.
Figure 1. HPLC data of a mycosporine glycine biosynthesis pathway. The DA construct is shown as a negative control, lacking mycosporine glycine and 4-deoxygadusol production. Clear peaks for these compounds are observed in the DOA sample.
Balskus and Walsh, 2010. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116657/]
4-DG Pathway (Optimised for E.coli)
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.
ATPG and NRPS Composite (Optimised for E.coli)
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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 250
Illegal EcoRI site found at 2397
Illegal XbaI site found at 3468 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 250
Illegal EcoRI site found at 2397
Illegal NotI site found at 1373
Illegal NotI site found at 2970
Illegal NotI site found at 4030 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 250
Illegal EcoRI site found at 2397
Illegal BglII site found at 134
Illegal BglII site found at 1587
Illegal BglII site found at 3184
Illegal XhoI site found at 1381
Illegal XhoI site found at 2978
Illegal XhoI site found at 4038 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 250
Illegal EcoRI site found at 2397
Illegal XbaI site found at 3468 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 250
Illegal EcoRI site found at 2397
Illegal XbaI site found at 3468
Illegal AgeI site found at 3568 - 1000COMPATIBLE WITH RFC[1000]