Part:BBa_K3634005:Design
ATPG and NRPS Composite (E.coli Optimised)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1561
Illegal NheI site found at 1584 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2059
Illegal BamHI site found at 4112 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Due to the two-phase nature of our project, the selected promoter and RBS used may be subject to change after wet lab application in 2021 to maximise shinorine production. Golden gate assembly methods will facilitate this optimisation if necessary. It may also be required to include more than one single promoter for adequate expression of NRPS compared to the other enzymes involved in the shinorine pathway. It is also worth noting that the third enzyme (ATPG) may be included on plasmid A with NRPS instead of on plasmid B such that mycosporine glycine, which also absorbs UV radiation to a certain degree, is not produced in a bacteria which by chance has taken up plasmid B and would thereafter exploit UV resistance to confer a survival advantage in the environment.
Source
The initial genomic sequence came from Anabaena variabilis ATCC 29413 which can be obtained from part K8140003 (Minnesota iGEM, 2012) before being optimised using the IDT optimisation tool. The promoter and RBS were selected from each respective Anderson catalogue based on their relative strength. The terminator BBa_B0015 sequence can also be taken from the iGEM registry.