Part:BBa_K3279006

INP-N ( N-terminus of Ice-nucleation protein ) from Pseudomonas syringae

Ice-nucleation protein (INP) is a secreted outer membrane protein, which is widely distributed in Pseudomonas syringae, Pseudomonas fluorescens and other Gram-negative bacteria. Compared with other surface carrier proteins, ice-nucleation protein has the advantage of stably expressing heterogenous proteins and displaying proteins with larger molecular weight. This part includes the sequence of INP-N (N terminus of Ice-nucleation protein), which is much shorter than INP and INP-NC. To allow this part fusion with other protein, we added an additional linker sequence at the 3'.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 333
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 75
  Illegal NgoMIV site found at 408
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization from iGEM19_CAU_China

With INP’s impressive ability, we fused the cellulases with INP-N to accomplish the purpose: cellulose degradation on the surface of bacteria. We linked the cellulases (cex and cenA seperately) into a pET30a(+) backbone, then transformed this plasmid into BL21. We induced these recombinant E.coli strains overnight under the condition of 16℃ 0.08 mM. See Fig.1[1].

To confirm whether it worked or not, we first detected the presence of the target protein by immunofluorescence staining and compared the fluorescence pattern and compared it with a negative control where cellulases was not fused with INP. We attached the His-tag to the fusion protein, so that it could allow an anti-His-tag primary antibody to combine the fusion protein and then let a fluorescence secondary antibody recognize them. As the result, we could spot the fluorescence of GFP when INP-N was fused with cellulase. See Fig.2[1].

Then the effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as substrate. See Fig.3[1].