Part:BBa_K4653106
Flagellin [Pseudomonas aeruginosa]
Flagellin is a globular protein that arranges itself in a hollow cylinder to form the filament in a bacterial flagellum. It has 161 amino acids. Flagellin is the principal component of bacterial flagella, and is present in large amounts on nearly all flagellated bacteria.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 289
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 28
Illegal PstI site found at 289 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 289
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 289
- 1000COMPATIBLE WITH RFC[1000]
Biology
The full length flagellin from Pseudomonas aeruginosa.The 22-amino acid sequence (flg22) of the conserved N-terminal part of flagellin is known to activate plant defence mechanisms. Flagellin perception in Arabidopsis thaliana functions via the receptor-like-kinase FLS2 (FLAGELLIN SENSING 2). Upon flg22 detection, FLS2 quickly binds to BAK1 (BRI1-associated kinase 1) to initiate signalling by reciprocal transphosphorylation of their kinase domains. Mitogen-activated-protein-kinases (MAPK) acts as downstream signalling compounds, leading ultimately to PAMP-triggered immunity in which more than 900 genes are up-/down-regulated upon flg22 treatment.
Design
We blasted the amino acid sequence of Flg22 at NCBI, screened the amino acid sequence of full-length flagellin isolated 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 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 full length Flagellin was induced by IPTG.
Functional verification
In the experiment, we tried to induce the expression of full length Flagellin by E. coli, then treat plant leaves with full length Flagellin, 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 full length Flagellin 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. Flagellin 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 full length Flagellin 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
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