Part:BBa_K3482037

Azurin with NSP4 secretion and 3XFLAG tag

Truncated azurin fused with a 3XFLAG tag and with a NSP4 secretion signal peptide for protein expression and purification

Usage and Biology

Azurin is a blue copper-binding redox protein originally found in the plasma membrane of Pseudomonas aeruginosa. It has recently been discovered to have an l anticancer effect by targeting the well-known p53 apoptosis pathway and by preferentially entering cancerous cells.

We used this part to test if azurin was produced in our bacteria.

Characterization

We fused a widely used 3XFLAG tag at the C-terminus of our azurin gene (BBa_3482040). This allowed us to perform a Western Blot, which allowed us to confirm the presence or absence of our expressed azurin. In addition, we modified the existing part BBa_K2500001 (truncated azurin uploaded by the ETH Zürich team of 2017) with either of the secretion tags pelB or NSP4[4] and investigated their secretion capability. We used an E. coli Nissle 1917 strain, which as been transformed with a modified sponge plasmid (usable in combination with a repressilator plasmid in the future) which contains our target genes constitutively expressed under a pTet promoter (BBa_K3482040, BBa_K3482041, BBa_K3482038, and BBa_K3482037) to performe a Western Blot with overnight cultures set to an OD600 of 2.0. The culture supernatant and lysate (pellet) were analyzed.

Western blot analysis of FLAG-tagged azurin from 4 different parts, showing two types of samples: supernatants or cell lysate. Immunoblotting after SDS-PAGE migration, membrane transfer, and analysis performed with 3XFLAG antibody. Samples are as follow: 1: BBa_K3482040 = azurin-3XFLAG; 2: BBa_K3482041 = truncated azurin-3XFLAG; 3: BBa_K3482038 = pelB-5D-truncated azurin-3XFLAG; 4: BBa_K3482037= NSP4-truncated azurin-3XFLAG.

We can observe the presence of the FLAG peptide in all cell lysate extracts. In addition, we can see that the full-length azurin as well as the 2 versions bearing a secretion tag (pelB or NSP4)(samples 3 and 4) are present in the supernatant. This leads us to conclude that our azurin is indeed expressed and secreted.

Sequence and Features

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 48
  
• 1000
  COMPATIBLE WITH RFC[1000]