Part:BBa_K3989010
plant immune elicitor flg22 fused with ClyA for OMV display
In this construct, outer membrane-associated protein ClyA (Part:BBa_K811000) is used to display the plant immune elicitor flg22 (Part:BBa_K3286138) on the surface of bacterial outermembrane vesicles (OMVs) to trigger plant immune responses. To make flg22 better recognized by plant immune receptor FLS2, we put mutiple linkers and a 3×FLAG tag between ClyA and flg22 to minimize the steric hindrance.
By doing western blot using anti-FLAG antibody, we proved that ClyA-elicitor fusion proteins indeed exist in OMVs (Figure 1).
Figure 1. ClyA protein with or without elicitor fused exists in outer membrane vesicles of E. coli<i> omp8 strain.
The immunogenicity of engineered OMVs was tested by Seedling Growth Inhibition (SGI) assay (Figure 2 & 3）, in which lower seedling weight indicates that higher immune responses are triggered (due to the trade-off between immunity and growth). Two OMV-producing <i>E. coli<i> strains were used, omp8 and <i>tolB<i>. In both strain, ClyA-flg22 and ClyA-elf18 can trigger stronger immune responses.
Figure 2.
Figure 3.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1105
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1105
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1105
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1105
Illegal NgoMIV site found at 1099
Illegal AgeI site found at 1063 - 1000COMPATIBLE WITH RFC[1000]
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