Part:BBa_K4195133

Biology

INPNC

INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C-terminal domains. It is a membrane protein commonly used to display protein of interest on the cell surface (1).

rLvAPN1

LvAPN1, a protein from the aminopeptidase N family, was identified in Litopenaeus vannamei hemocytes as a receptor for VP_AHPND toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (2).
rLvAPN1 is a truncated form of LvAPN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins (2). What’s more, there is no glycosylation site in rLvAPN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as E. coli).

Usage and design

Engineering outer membrane vesicles (OMVs) for treating and preventing AHPND caused by the pathogen V. parahaemolyticus are a significant part of OMEGA project (Operable Magic to Efficiently Getting over AHPND). Based on the efforts of our previous projects in 2020 (AnTea-Glyphosate) and 2021 (SALVAGE), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins was no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), rLvAPN1 (receptor), TTPA (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by V. parahaemolyticus. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems which we have been dedicated to since 2020. Learn more information from our Design page.

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
This composite part was constructed at pSB1C3 to express INPNC-rLvAPN1-his (BBa_K4195032), which is induced by L-arabinose. We transformed the plasmid into E. coli BL21(DE3) for further verification of its location on the surface of E. coli.

Characterization

1.Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3900 bp) can be observed at the position between 3000 and 5000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195133_pSB1C3.

2. Characterization of displaying cargo proteins

We used BBa_K4195134 which has no surface display system (like INPNC or ClyA) as negative control and BBa_K4195133 as positive control. The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody to verify whether the display system is functional or not.

Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0406).
The ratio of fluorescence intensity (_λEx = 492 nm, λ_Em = 528 nm) to OD₆₀₀ of positive control (INPNC-rLvAPN1-his) is higher than that of negative control (rLvAPN1-his) (Fig. 3), which indicates that our surface display system works well that the receptor rLvAPN1 is successfully located on the surface of bacteria.

Reference

1.E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechno. 29, 79-86 (2011).
2.W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  Illegal NheI site found at 1705
  Illegal NheI site found at 3388
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1308
  Illegal NgoMIV site found at 1918
  Illegal NgoMIV site found at 3027
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1665
  Illegal AgeI site found at 2059
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1375
  Illegal SapI site found at 961
  Illegal SapI site found at 3392