Difference between revisions of "Part:BBa K4653103"

Revision as of 12:46, 12 October 2023

3xFlg22

Flg22 a 22-amino acid sequence from the N-terminal of flagellin from P. aeruginosa, has been shown to be a pathogen-associated molecular model PAMP, capable of binding to FLS2 receptors on the surface of plant cells and stimulating plant immunity.We repeated the sequence of Flg22 three times to increase the probability of our PAMP reaching the plant pattern recognition receptor to enhance the plant immune response.

Sequence and Features

Biology

Flg22 is a conserved flagellin short peptide containing only 22 amino acid sequences. Flg22 comes from P. aeruginosa, a less pathogenic but highly drug-resistant bacterium. Widely existing in nature, it is a common bacterial infection in wound. Plant cells have the flagellate-sensitive receptor FLS2, a pattern recognition receptor located on the cell surface that recognizes some common, conserved molecular motifs on the pathogen and triggers an innate immune response, namely PTI. When flg22 activates the plant cell surface receptor FLS2, it will induce its co-receptor BAK1 to act together, activate downstream including plant hormone SA pathway, MAPK cascade pathway, reactive oxygen species outbreak and enhance plant disease resistance.
In order to achieve better immune effect, the sequence of Flg22 was repeated three times, and the flexible amino acid sequence was linked to make the expressed protein have more binding sites with the pattern recognition receptor FLS2, so as to enhance the plant immune strength.

Design

We blasted the amino acid sequence of Flg22 at NCBI, found the homologous sequence from P. aeruginosa, and then selected it as the basis for back translation and codon optimization. Sequence optimization was carried out for E. coli expression, resulting in an optimized GC level of 0.59, reducing the occurrence of rare codons and avoiding hairpin structure. The cutting sites of Bsa I and Bbs I were deleted to meet the requirements of Biobrick, and then the stop codon was introduced.
In order to construct the repeat fragment of Flg22 truncated sequence, GGSGG was selected as a flexible linker to obtain the amino acid sequence of 3x repeated Flg22 truncated sequence（QRLSTGSRINSAKDDAAGLQIAGGSGGQRLSTGSRINSAKDDAAGLQIAGGSGGQRLSTGSRINSAKDDAAGLQIA）

In terms of experimental operation, we hope that the expressed protein can have good water solubility and be easy to purify, so we hope to use calculation tools based on protein solubility weighted index to predict the influence of several commonly used protein affinity labels on protein solubility. Finally, we choose SUMO label and His label to connect proteins. Help to improve its expression and subsequent purification process.

Plasmid construction

Our combined fragment was recombined into pET-28a(+) vector, and the recombinant plasmid was transferred into proteinase-deficient Escherichia coli BL21 (DE3), and the fermentation production of 3×Flg22 was induced by IPTG.

Functional verification

In the experiment, we tried to induce the expression of Flg22 by Escherichia coli, then treat plant leaves with 3×Flg22, and immunoresponse-related indicators were then tested to verify effectiveness. However, due to the short experimental time and limited equipment conditions, we gradually gave up the program, but we still chose to record our experimental attempts to provide experience for iGEM teams who choose plant immune-related content or use 3×Flg22 in the future.

Recombinant plasmid electrophoresis

In our experiment, the target protein was extracted and purified according to the protein label and treated to the leaves of tomato. BvEP can stimulate the outbreak of reactive oxygen species in tomato leaves as an immune response. Hydrogen peroxide, a kind of ROS, can be used to calculate or predict the degree of immune response by observing or counting the relative gray value of leaves after the brown red precipitates generated by DAB staining are decolorized by ethanol.

Expression of Proteins

The expression of immune protein factors is regulated by lactose operon, so IPTG is applied to induce our protein expression, and His labeled protein purification kit is used to extract and purify Flg22 protein. After extracting the protein, the protein concentration was determined by BCA method. We controlled the experiment, and the protein expression level was low when induced at 100 rpm in a shaker at 25℃. We also tried different conditions later, and it did not exceed 0.2mg/mL.

Due to the short experimental time and limited equipment conditions, we gradually gave up the oligosaccharide production program, but we still chose to record our experimental attempts to provide experience for iGEM teams who choose plant immune-related content in the future.

Reference

[1] Chinchilla, D., Zipfel, C., Robatzek, S. et al. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500 (2007).doi.org/10.1038/nature05999.
[2] Denoux C, Galletti R, Mammarella N, et al. Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings[J]. Molecular plant, 2008, 1(3): 423-445.
[3] Orosa, B., Yates, G., Verma, V. et al. SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity. Nat Commun 9, 5185 (2018). https://doi.org/10.1038/s41467-018-07696-8.
[4] Jelenska J, Davern SM, Standaert RF, Mirzadeh S, Greenberg JT. Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2. J Exp Bot. 2017 Mar 1;68(7):1769-1783. doi: 10.1093/jxb/erx060.
[5] Sun Y, Li L, Macho A P, et al. Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex[J]. Science, 2013, 342(6158): 624-628.
[6] Chinchilla D, Bauer Z, Regenass M, et al. The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception[J]. The Plant Cell, 2006, 18(2): 465-476.
[7] Yi S Y, Shirasu K, Moon J S, et al. The activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition[J]. PloS one, 2014, 9(2): e88951.
[8] Annabelle Decreux, Johan Messiaen, Wall-associated Kinase WAK1 Interacts with Cell Wall Pectins in a Calcium-induced Conformation, Plant and Cell Physiology, Volume 46, Issue 2, February 2005, Pages 268–278, https://doi.org/10.1093/pcp/pci026