Part:BBa_K4195010

clyA-ttpB

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.

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

For this part (ClyA-TTPB), TTPB 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 composite part BBa_K4195103. 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 (4670 bp) can be observed at the position around 5000 bp (Fig. 2).

Fig.2 DNA gel electrophoresis of the colony PCR products of BBa_K4195103_pSB1C3.

2. Ability of binding Vp0980 on the surface of engineered bacteria

We used BBa_I0500 promoter and RBS (BBa_B0034) to express ClyA-TTPB protein in E. coli BL21(DE3). The arabinose-induced overnight culture was then incubated with purified Vp0980-his and FITC-labeled anti-His-tag antibody in turn to verify whether the displayed TTPB could bind Vp0980 or not.

3. Ability of binding Vp0980 on the surface of OMVs

For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195121 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified Vp0980-his and anti-His-tag antibody in turn and finally probed by the HRP-conjugated secondary antibody (3, 4). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed TTPB on OMVs is functional or not.

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).

3. J. L. Valentine et al., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. Cell Chem Biol 23, 655-665 (2016).

4. T. C. Stevenson et al., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. Proc Natl Acad Sci U S A 115, E3106-E3115 (2018).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 325
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2166
  Illegal BamHI site found at 2088
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1210
  Illegal AgeI site found at 2638
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 772