Composite

Part:BBa_K4195101

Designed by: Xiaoping Yu   Group: iGEM22_XMU-China   (2022-09-27)


I0500-B0034-clyA-ttpB-his-B0015


Biology

ClyA

Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family. When fused to the C-terminal of ClyA, heterologous proteins can be displayed on the surface of the engineered bacteria and OMVs (outer membrane vesicles) released by them (1).

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 (2).

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), TTPB (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.

T--XMU-China--surface display circuit.png

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.

TTPB-his was fused to the C-terminal of ClyA to surface display for targeting V. parahaemolyticus. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then constructed this part. 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).

T--XMU-China--BBa K4195101（ClyA-ttpB-his,colony PCR,BL21(DE3)).png

Fig.2 DNA gel electrophoresis of the colony PCR products of BBa_K4195101_pSB1C3.

2. Characterization of displaying cargo proteins

We used BBa_I0500 promoter and RBS (BBa_B0034) to express ClyA-TTPB-his protein in E. coli BL21(DE3). The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody in turn to verify whether the TTPB was displayed on the surface of E. coli or not.

T--XMU-China--CtB.png

Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0019).

The ratio of fluorescence intensity (λEx = 492 nm, λEm = 518 nm) to OD600 of positive control (E. coli harboring BBa_K4195101) is higher than that of negative control (E. coli harboring BBa_K4195008) (Fig. 3), which indicates that our surface display system can successfully display TTPB on the surface of E. coli.

Reference

1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel). 14, 78 (2022).

2. 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 1408
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3402
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1783
    Illegal NgoMIV site found at 1795
    Illegal NgoMIV site found at 2955
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2866
    Illegal AgeI site found at 3214
    Illegal AgeI site found at 3280
    Illegal AgeI site found at 3522
    Illegal AgeI site found at 3874
    Illegal AgeI site found at 4060
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 3199


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