Difference between revisions of "Part:BBa K2450401:Design"
 
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===Design Notes===
 
===Design Notes===
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The spytag has been placed on the N terminal of the sfGFP. This is because we were unsure if the spytag/spycatcher isopeptide bond would still be formed if the spytag was in the centre of the protein. However, we were unsure if this design would affect the insertion of OmpA into the membrane. An alternative part [BBa_K2450451] with the opposite design has been created so the spytag is in the centre of the protein, so we are more confident in the insertion of OmpA into the membrane but less confident in the isopeptide bond formation.
+
The spytag has been placed on the N terminal of the OmpA. This is because we were unsure if the spytag/spycatcher isopeptide bond would still be formed if the spytag was in the centre of the protein. However, we were unsure if this design would affect the insertion of OmpA into the membrane. An alternative part [BBa_K2450451] with the opposite design has been created so the spytag is in the centre of the protein, so we are more confident in the insertion of OmpA into the membrane but less confident in the isopeptide bond formation.
  
 
sfGFP rather than GFP has been used as this part is being targeted to the outer membrane, and GFP cannot fold in the periplasmic space.  
 
sfGFP rather than GFP has been used as this part is being targeted to the outer membrane, and GFP cannot fold in the periplasmic space.  
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===References===
 
===References===
 +
Alves, N. J., Turner, K. B., Medintz, I. L., & Walper, S. A. (2016). Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles. Scientific Reports, 6(1), 24866. http://doi.org/10.1038/srep24866

Latest revision as of 12:51, 21 October 2017


OmpA SpyTag sfGFP label


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The spytag has been placed on the N terminal of the OmpA. This is because we were unsure if the spytag/spycatcher isopeptide bond would still be formed if the spytag was in the centre of the protein. However, we were unsure if this design would affect the insertion of OmpA into the membrane. An alternative part [BBa_K2450451] with the opposite design has been created so the spytag is in the centre of the protein, so we are more confident in the insertion of OmpA into the membrane but less confident in the isopeptide bond formation.

sfGFP rather than GFP has been used as this part is being targeted to the outer membrane, and GFP cannot fold in the periplasmic space.


Source

tba

References

Alves, N. J., Turner, K. B., Medintz, I. L., & Walper, S. A. (2016). Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles. Scientific Reports, 6(1), 24866. http://doi.org/10.1038/srep24866