Part:BBa_K2623021

Bacterial outer membrane protein A (OmpA) fused with SpyTag at its N-termini and GFP at its C-termin

Summary

This part is a relatively basic part designed to target the archaeal ribosomal protein protein L7Ae and then siRNA into outer-membrane vesicles (OMVs). Porin outer-membrane protein A, OmpA, is a kind of highly expressed transmembrane porin protein of E. coli and is therefore abundant in OMVs. We inserted SpyTag to its N-termini to bioconjugate with the SpyCather in the following parts. In order to test the efficiency of anchoring the OmpA-SpyTag (OmpA-ST) in our circuit, we use GFP, BBa_I13401 as the reporter, because the fluorescence intensity is easier to be measured.

Usage and Biology

As mentioned above, SpyTag was constructed in order to anchore L7Ae to outer membrane through the bioconjugation of the SpyTag/SpyCatcher system. We also construct another form of BBa_K2623021-BBa_K2623023. The only difference is that we inserted SpyTag to C-termini of OmpA protein(instead of the N-termi in BBa_K2623021). Through the fluorescence intensity of GFP, we can compare and evaluate expression efficiency of SpyTag between the BBa_K2623021 and this part. Then the better one would be uesd to construct our final gene circuit.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 65
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1302