Difference between revisions of "Part:BBa K2319005:Design"

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===Design Notes===
 
===Design Notes===
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<p><a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a> (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!</p>
 
<p><a href="https://parts.igem.org/Part:BBa_J18932">BBa_J18932</a> (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!</p>
  
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<p>By exploiting a natural NdeI site (CA\TATG) occurring right after the modified sequence, we insert <a href="https://parts.igem.org/Part:BBa_K2319006">BBa_K2319006</a> comprising a HindIII site (for insertion into the T7 expression backbone), a start codon (ATG), and a 6xHis-tag (for easy Ni-NTA column-based protein purification), and the modified sequence preceding the hidden translation start site.</p>
 
<p>By exploiting a natural NdeI site (CA\TATG) occurring right after the modified sequence, we insert <a href="https://parts.igem.org/Part:BBa_K2319006">BBa_K2319006</a> comprising a HindIII site (for insertion into the T7 expression backbone), a start codon (ATG), and a 6xHis-tag (for easy Ni-NTA column-based protein purification), and the modified sequence preceding the hidden translation start site.</p>
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===Source===
 
===Source===

Revision as of 10:21, 1 November 2017


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 First 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

By exploiting a natural NdeI site (CA\TATG) occurring right after the modified sequence, we insert BBa_K2319006 comprising a HindIII site (for insertion into the T7 expression backbone), a start codon (ATG), and a 6xHis-tag (for easy Ni-NTA column-based protein purification), and the modified sequence preceding the hidden translation start site.

Source

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

References