Results | SJTU-BioX-Shanghai

The engineered OMVs were collected using ultracentrifugation (see Methods) and characterized by Nanoparticle Tracking Analysis (NTA) (Fig 1A). The Pb²⁺ binding protein ClyA-PbrR-His6 (CPH) was successfully displayed on the surface of the OMVs (Fig 1B). The Pb²⁺ removal ratio was significantly higher in groups using CPH⁺ OMVs than in those using CPH^- OMVs (Fig. 2), indicating that the CPH protein enhanced the Pb²⁺ binding capability of the OMVs, yet the removal efficiency is strongly dependent on the OMV concentration. In conclusion, the Pb²⁺-binding function of CPH was verified.

(A) Size distribution graph of nanoparticles collected from ultracentrifugation. The peak value is within the range of 100-150 nm. (B) CPH is detected both in the crude extraction of bacterial cell total protein and in the OMVs. CPH, ClyA-PbrR-His6 protein; Ctrl, CPH^- OMVs; Test, CPH⁺ OMVs.

The Pb²⁺ removal ratio of the test group and the control group. (p < 0.01, **) CPH, ClyA-PbrR-His6 protein; Ctrl, CPH^- OMVs; Test, CPH⁺ OMVs.

To collect the Pb²⁺-absorbed OMVs, we first tested an Lpp-OmpA-His10 protein construct on OMVs’ surface to endow it with Ni-binding affinity.. The Lpp-OmpA-His10+ OMVs were detected in the effluent of both 25 mM and 500 mM imidazole elution buffer (data not shown), which demonstrated that the Lpp-OmpA-His10 protein enabled the OMVs to be ‘caught’ by the Ni beads. The surface-displayed His10-tag was proved to be functional despite its wide-ranging binding strength.

(A) Western Blot result demonstrating the presence of Lpp-OmpA-His10 protein in different OMV suspensions collected throughout the Ni-binding experiment. The control group consisted of OMVs without engineering. Group a and b are parallel replications. (B) Column graph showing the recovery rates for His-Tag^- and His-Tag⁺ OMVs. The statistical analysis is performed on the sum of IMZ-25 and IMZ-500. IMZ-25, OMVs eluted by 25 mM Imidazole solution; IMZ-500, OMVs eluted by 500 mM Imidazole solution. (p < 0.01, **)

To combine the Pb²⁺- and Ni-binding functions into one single part, a His10-tag with a GGGGS linker was added at the end of CPH (see Design), forming the elongated version of the heavy metal binding module, ClyA-PbrR-GGGGS-His10 (the Elongated version, “Elo”). The Ni binding performance of Elo⁺ OMVs was similar to that of Lpp-OmpA-His10⁺ OMVs, which was statistically higher than that of empty OMVs vesicles (Fig. 3B). Similarly, Elo⁺ OMVs captured by Ni beads showed varied affinity (Fig. 3A).

The OMVs displaying SpyTag(ST) and SpyCatcher(SC) proteins were successfully produced respectively by two E. coli strains, collected by ultracentrifuge, verified by Western Blotting, and measured by Nanoparticle Tracking Analysis (NTA). The ST⁺ OMVs and SC⁺ OMVs were mixed and allowed to generate OMV clusters at room temperature (Fig. 4A). Compared with ST⁺ and SC⁺ OMV suspension, there were more bright and large particles twinkling in the mixture, indicating that the ST⁺ and SC⁺ OMVs had aggregated to form clusters (Fig. 4B-D).

(A)The schematic diagram of SC⁺ OMV (in red) and ST⁺ OMV (in blue) undergoing crosslink. (B)Nanoparticle Tracking Analysis (NTA) of ST+OMVs. (C)NTA of SC⁺ OMVs. (D)NTA of crosslinking of ST⁺ OMVs and SC⁺ OMVs.The ST⁺/SC⁺ OMV clusters are indicated by the red arrow. SC⁺ OMV, OMV with SpyCatcher; ST⁺ OMV, OMV with SpyTag; ST-SC⁺ OMV, OMV groups formed by ST⁺ OMV and SC⁺ OMV.

To more clearly demonstrate the differences before and after the cross-linking reaction, quantitative analysis was performed to better characterize the formation of OMV aggregates (Fig. 5A). By focusing on the particles with greater diameters, some unique size distribution features were discovered. There were a few distribution peaks located at large diameter values in the mixture, which were absent in either ST⁺ or SC⁺ OMVs (Fig. 5B,5D), suggesting the presence of cross-linking OMV clusters. Also, the single-OMV-sized particles were fewer in the mixture than in the other two inputs (Fig. 5C), which could be explained by the OMV-consuming process of aggregation.

(A-B) Counting of OMVs of different sizes in the three groups of OMV suspensions revealed that there were many populations of OMVs of larger sizes (Diameter > 400 nm) in ST⁺/SC⁺, as shown by dashed boxes in Fig. A and arrows in Fig. B. (C-D) The proportions of different sized OMVs to the total number of OMVs in the three sets of OMV suspensions are counted. (C) The proportion of smaller OMVs (Diameter < 220 nm). (D) The proportion of larger OMVs (Diameter> 500 nm).ST⁺, OMV with SpyTag; SC⁺, OMV with SpyTag; ST⁺/SC⁺, OMV groups formed by ST and SC.

The cross-linking function of anchored ST and SC was then confirmed by the Western Blotting assay of the cross-linking products. The ligated protein, ST-SC, was expected to stably exist in the mixture since the two proteins were linked by a covalent bond (Fig. 6A). A larger positive band was detected with antibodies targeted at both ST and SC with the same pattern throughout the different mixture groups (Fig. 6B-C). The presence of the bands was determined by the simultaneous existence of ST⁺ and SC⁺ OMVs, while the brightness of some bands was influenced by the unstable yield of SC⁺ OMVs (Fig. 6D).

(A) SpyTag and SpyCatcher 3D structure diagrams and the schematic diagram of the crosslinking of SpyTag and SpyCatcher. (B-C) Western Blot results demonstrated that some groups of the mixture of ST⁺/SC⁺ OMV suspensions had both SpyTag and SpyCather. The control group consisted of OMVs with ST or SC only. (D) Qualitative comparison of ST, SC, and ST-SC levels in each group in Western Blot experiments in Fig.B and Fig.C. ‘+’: Protein is present; ‘-’: Protein is not present; The number of ‘+’ represents the relative expression level of each protein observed by the eye. ST, SpyTag; SC, SpyTag; ST-SC, ligated SpyTag and SpyCatcher; M1-M8, the mixture of ST+ and SC+ OMV.

In conclusion, the engineered OMVs successfully performed Pb²⁺ binding, Ni binding, and cross-linking functions. The OMV system that SJTU-BioX-Shanghai designed this year to remove Pb²⁺ from the waters has accomplished the proof of concept.