Coding
Intein

Part:BBa_K1483003:Design

Designed by: Nikolas Layer, Philipp H. O. Mayer and Philip Roessler   Group: iGEM14_Tuebingen   (2014-10-06)
Revision as of 21:41, 14 October 2014 by Philip (Talk | contribs) (→‎Source)

Ssp GyrB Split Intein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Part was designed in RFC25, since it is specifically used for fusion of peptides and proteins. The codon usage was modified for expression in E. coli K12. Common restriction sites were removed. The gene was obtained by in-vitro gene synthesis.

The complementary C-intein was generated by solid phase peptide synthesis. The functional part of the C-intein was extended c-terminaly by a linker consisting of aminocaproic acid (also knwon as ε-Ahx), L-lysine and L-cysteine. The lysine was coupled to carboxyfluorescein to allow detection of the peptide and thereby enable labeling of intein-fused proteins. The synthetic peptide can be immobilised on sulfo link beads using the cystein.

SynPeptideTuebingen1.jpg


Source

Intein of DNA gyrase subunit B of the cyanobacterium Synechocystis spec. PCC6804. The sequence was optimized for codon usage in E. coli K12 and obtained by in-vitro gene synthesis.

References

The sequence of the intein protein can be found in the [http://tools.neb.com/inbase/intein.php?name=Ssp+GyrB Intein Database and Registry]