Part:BBa_K5184052
HrpN
In order to rejuvenate cultivations infected by spider mites, HrpN is incorporated in our project with reparation and improvement means. Harpins are thermally stable proteins produced by gram-negative plant pathogenic bacteria. Once applied exogenously to cultivations, several beneficial responses could be induced including systemic acquired resistance, hypersensitive response effect (HR) and plant growth via stimulating an increase in root and shoot biomasses. Likewise, harpin proteins are competent to enhance plant disease resistance via mutual coordination and plant defense response against diverse pathogens and insects. As an omnipotent treatment for plants in all conditions, HrpN provides future iGEM teams dedicate in incorporations of plant health intervention strategies an appealing and effective choice.
Essential Information
Sequences
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
HrpN, first found in Erwinia amylovora, contain several α-helical regions and have an acidic isoelectric point (pI). Once applied exogenously, it is capable of triggering the hypersensitive response in plants, characterized by the rapid death of plant cells at the site of pathogen invasion. This response is a defense mechanism to limit pathogen spread,. Harpins activate various plant defense pathways, including the production of reactive oxygen species (ROS) and the expression of defense-related genes. Beyond local responses, harpins can enhance systemic acquired resistance, a mechanism of induced defense that confers long-lasting protection against a broad spectrum of microorganisms, thus leading to an enduring resistant to a broad range of pathogens.
Characterization
A research [13] suggests that the original E. amylovora and the codon optimized version (for E. coli) of the harpin protein gene leads to formation of secondary structures in its translation initiation region (TIR) of its mRNA and impacts final protein yield. hrpN is the TIR-optimized version, with a less negative delta G for secondary structure formation in its TIR of the original coding sequence comparing with the unoptimized version, hrpN-ori.
Plasmid for synthesis of hrpN is transformed into BL21(DE3), cultured under 37°C, and is subsequently induced with IPTG when OD600 reached 0.8-1.0. Supernatant and precipitate were collected after sonication cell lysis.
The supernatant was purified via 6×His tag, followed by dialyzing the purification product. The results were assessed via SDS-PAGE [Fig2A], revealing successful expression of all proteins. A BCA assay was carried out then to determine specific protein yield [Fig2B], [Fig2C].
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