Part:BBa_K4195033
clyA-his
Biology
ClyA Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family. When fused with 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).
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 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.
In order to purify OMVs (2), we added a his-tag (6×His) at the C-terminal of ClyA. We used both BBa_I0500 and BBa_B0034 to construct the expression system and obtained the composite part BBa_K4195135, which are assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
Characterization
1. Identification
After transforming the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (2662 bp) can be observed at the position between 3000 bp and 2000 bp (Fig. 2).
Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195135_pSB1C3.
2. Immunofluorescence (IF)
The culture was incubated overnight. Then the FITC-labeled anti-His-Tag antibody was used to target the His-tag (6×His) displayed via ClyA, followed by measuring the fluorescence intensity (λEx= 492 nm, λEm = 528 nm) to OD600 of the culture.
The results showed that the ratio of fluorescence intensity to OD600 of positive control (bacteria harboring BBa_K4195135) is higher than that of negative control (bacteria harboring BBa_K4195119) (Fig. 3), which indicated that these two surface display systems can work well.
Fig. 3 Fluorescence intensity/OD600 of E. coli whether express the anchor protein or not (p=0.0003).
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. N. J. Alves, K. B. Turner, K. A. DiVito, M. A. Daniele, S. A. Walper, Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant. Res Microbiol 168, 139-146 (2017).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 172
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 772
Illegal SapI site found at 297
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