Difference between revisions of "Part:BBa K2319007:Design"

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===References===
 
===References===
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RBS Calculator: https://salislab.net/software/

Latest revision as of 01:19, 2 November 2017


improved mCherry RFP


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

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 modified from BBa_J18932 using an oligo to attach the modified sequence using an NdeI site.

References

RBS Calculator: https://salislab.net/software/