Part:BBa_K3989014

double cellulose binding domain fused with ClyA for OMV display

In this construct, outer membrane-associated protein ClyA (Part:BBa_K811000) is used to display the double cellulose-binding domain (dCBD, Part:BBa_K1321340) to the surface of bacterial outer membrane vesicles (OMVs). To make dCBD better displayed, we put multiple linkers and a 3×FLAG tag between ClyA and dCBD to minimize the steric hindrance.

Usage and Biology

This construct can be used to enhance the plant immune responses triggered by OMVs, please see: 2021 UZurich

With the cellulose-binding domain displayed on the OMV surface, recovery of the engineered OMVs could be achieved easily via cellulose pull-down (Su et al., 2017).

Characterization by team 2021 UZurich

Seedling growth inhibition (SGI) assay was performed to test whether displaying cellulose-binding domain on OMV surface can enhance the plant immune response (the lower the seedling weight, the stronger the plant immune response). OMV with only ClyA (blue boxes) was used as the negative control.

As the figure shown below, the seedling weight of the negative control group gradually decreases as OMV concentration increases. However, for ClyA-dCBD, the seedling weight dramatically decreases between concentrations of 0 and 2.5, but doesn't decrease a lot as concentration further increases, which means the immune response might get saturated.

One possible explanation of such results is the enrichment of OMVs by cellulose in the cell wall. At low concentration, the cell wall limits the diffusion of wild-type OMVs, but for engineered OMVs with dCBD, they get enriched in the cell periphery, generating higher local concentration and facilitating the diffusion. At higher concentrations, the enrichment effect gets saturated, and OMVs attached to cellulose can even block free OMVs to enter, resulting in no difference between ClyA-dCBD and ClyA-only groups.

Reference

Su, F. H., Tabañag, I. D. F., Wu, C. Y., & Tsai, S. L. (2017). Decorating outer membrane vesicles with organophosphorus hydrolase and cellulose-binding domain for organophosphate pesticide degradation. Chemical Engineering Journal, 308, 1-7.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]