Part:BBa_K4195011
INPNC-ttpB
Biology
INPNC
INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C- terminal domains (1). It is a membrane protein commonly used to displayed protein on the cell surface (2).
TTPB
TTPB is tail tubular protein B of podophage 7. It has been found that TTPB serves as ligands that recognizes the conserved Vibrio receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of Vibrio parahaemolyticus and then mediates phage adsorption and subsequent bacterial lysis (3).
Usage and design
Engineering 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 were 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 Designpage.
Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
For this part (INPNC-TTPB), TTPB was fused to the C-terminal of INPNC to surface display for targeting V. parahaemolyticus. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then constructed composite part BBa_K4195102. We transformed the constructed plasmid into E. coli BL21(DE3) for further verification of its expression and function on the surface of E. coli and OMVs, including the interaction between TTPB and Vp0980.
Characterization
1. Identification
When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (4688 bp) can be observed at the position around 5000 bp (Fig. 2).
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Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195102_pSB1C3.
Reference
1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends. Biotechnol. 29, 79-86 (2011).
2. http://2016.igem.org/Team:TJUSLS_China.
3. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerg. Microbes. Infect. 9, 855-867 (2020).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 301
Illegal NheI site found at 337
Illegal NheI site found at 2473 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 330
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal NgoMIV site found at 1737
Illegal AgeI site found at 823
Illegal AgeI site found at 1093
Illegal AgeI site found at 1228
Illegal AgeI site found at 1648
Illegal AgeI site found at 1996
Illegal AgeI site found at 2827
Illegal AgeI site found at 3018 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1726
Illegal SapI site found at 944
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