Part:BBa_K5184053

HrpN

In order to rejuvenate cultivations infected by spider mites, hrpN-ori 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-ori provides future iGEM teams dedicate in incorporations of plant health intervention strategies an appealing and effective choice.

Essential Information

Sequences

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]

Usage and Biology

hrpN-ori, 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 [1] 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.

Figure 1: A. Harpin will induce hypersensitive response in plants; this will thus promote growth and a faster recovery of the plant after a spider mite infestation B. Plasmids constructs for hrpN and hrpN-ori C. RNA secondary structure prediction and their associated free energy changes in the TIR of hrpN-ori and hrpN using RNAfold

Plasmids for synthesis of hrpN and hrpN-ori are 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].

Figure 2: A. Purification of hrpN and hrpN-ori after bacterial harvest, with complete protein of BL21(DE3) as control S: supernatant, P: resuspended precipitate, FT: flowthrough, W: wash E: elusion B. BCA assay of hrpN and hrpN-ori C. Comparison of protein yields of hrpN(152.94mg/μl) and hrpN-ori(106.89mg/μl), statistical test: unpaired t-test suggests a significant difference ****: p< 0.0001

BCA assay suggests that the optimized hrpN results in a significant increase in protein yield comparing to hrpN-ori [Fig2C], signifying the effect of the structure of the TIR on expression rate and thus yield of the protein.

Reference

[1]Cai, Zengying, et al. ‘Efficient Expression and Purification of Soluble HarpinEa Protein by Translation Initiation Region Codon Optimization’. Protein Expression and Purification, vol. 188, Dec. 2021, p. 105970. DOI.org (Crossref), https://doi.org/10.1016/j.pep.2021.105970.