Part:BBa_K3634003:Design

Nonribosomal Peptide Synthetase (NRPS) (Codon Optimised for E.coli)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 458
  Illegal BamHI site found at 2511
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Initially, part BBa_K814003 (Minnesota iGEM, 2012) was taken and the sequence found to include a single XbaI illegal restriction site at bp 2604. The appropriate synonymous mutation was made in silico to remove this site (t2604a). This allowed for the part to be used at RFC[10] and RFC[1000] standard without codon optimisation. The sequence was then optimised using the IDT codon optimisation tool. This resulted in a new SapI, EcoR1 and PstI illegal restriction sites at bp 855, 1195 and 1976 respectively. The appropriate synonymous mutation was then made in silico to remove these additional sites (t852a, a1197g & g1974c). This allowed for the part to be used at RFC[10] and RFC[1000] standard with codon optimisation. Unlike previous parts optimised, the NRPS optimised sequence was flagged as being of ‘low complexity’ indicating that some complexities with the part existed but it was not anticipated to be problematic. One point of this described complexity was that one or more repeated sequences greater than 8 bases comprised 48% of the overall sequence. The second point of complexity was that a specific region of 13 bases was composed of 84.6% G bases, starting at bp 1111. It was suggested that if issues were to arise from the above part sequence, it be redesigned to reduce the repeats to less than 40% of the sequence. As for the GC rich region, the length of the sequence should be reduced to under 8 bases or the percentage of G base composition reduced to less than 80% respectively if any problems were to occur.

Source

The initial genomic sequence came from Anabaena variabilis ATCC 29413 which can be obtained from part BBa_K814001 (Minnesota iGEM, 2012). The part sequence was then optimised for our chosen chassis organism, E.coli, using the IDT codon optimisation tool.

References

Minnesota iGEM 2012 - https://parts.igem.org/Part:BBa_K814003

IDT Codon Optimisation Tool - https://eu.idtdna.com/CodonOpt

Balskus, E.P, Walsh, C.T. 2010. The Genetic and Molecular Basis for Sunscreen Biosynthesis in Cyanobacteria. Science. 329 (5999): p1653-1656. DOI: 10.1126/science.1193637