Generator

Part:BBa_K2319005:Design

Designed by: Raj Magesh Gauthaman   Group: iGEM17_IISc-Bangalore   (2017-10-31)
Revision as of 01:16, 2 November 2017 by Raj Magesh (Talk | contribs) (→‎Design Notes)


mCherry-SpyTag under T7 expression system


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 828
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 765
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

BBa_J18932 (mCherry RFP) has an interesting flaw: an internal ATG near the N-terminus has a RBS-like sequence preceding it; this hidden translation start site leads to ~50% truncation of the produced mCherry protein!

Our Modification — Improved mCherry

Using an in silico analysis of RBS strengths using an online RBS Calculator, we modified the nucleotide sequence preceding the translation start site to become a far weaker RBS while maintaining the same amino acid sequence. This inhibits translation initiation at that position by almost 75% (predicted) and so reduces the truncation of the protein.

BBa_J18932_mCherry      1 GTGAGCAAAGGCGAGGAAGATAACATG     27
                   |||...|||||.||.||||||||.|||
Improved_mCherry        1 GTGTCTAAAGGTGAAGAAGATAATATG     27

Source

This was assembled using existing BioBricks and some custom oligos to add specific sequences.

References