Difference between revisions of "Part:BBa K2959007"

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	===Characterization of Expressible Wheat antimicrobial peptide 1b===		===Characterization of Expressible Wheat antimicrobial peptide 1b===
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	Our DNA sequence (WAMP1b) was synthesized by IDT®️ with the Biobrick prefix and suffix flanking the composite part. This made possible the correct digestion with restriction enzymes EcoRI-HF and PstI. After the digestion, ligation  was performed with T7 ligase in order to place our construct into the pSB1C3 linearized backbone with chloramphenicol resistance, which was previously digested with the same restriction enzymes. Using the SnapGene®️ software, we could model our ligated expression plasmid, and the final part resulted in a sequence of  2,590 bp. Thereupon,<i>E. coli</i> SHuffle was transformed by heat shock for following antibiotic selection of clones.</p>		Our DNA sequence (WAMP1b) was synthesized by IDT®️ with the Biobrick prefix and suffix flanking the composite part. This made possible the correct digestion with restriction enzymes EcoRI-HF and PstI. After the digestion, ligation  was performed with T7 ligase in order to place our construct into the pSB1C3 linearized backbone with chloramphenicol resistance, which was previously digested with the same restriction enzymes. Using the SnapGene®️ software, we could model our ligated expression plasmid, and the final part resulted in a sequence of  2,590 bp. Thereupon,<i>E. coli</i> SHuffle was transformed by heat shock for following antibiotic selection of clones.</p>
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Revision as of 18:12, 19 October 2019

Expressible Wheat antimicrobial peptide 1b

This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for WAMP1b as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of WAMP1b, an antifungal peptide from Triticum kiharae seeds, in E. coli. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The part is designed to code for a fusion protein of WAMP1b with a polyhistidine tag (6x His-Tag) at its N-terminus for purification by immobilized metal affinity chromatography.

Usage and Biology

WAMP1b is an antimicrobial peptide from Triticum kiharae seeds. This peptide consists of 116 amino acids with a molecular weight of 11.5 kDa. Among these aminoacids, 10 cysteines are included, which form 5 nonconsecutive disulfide bonds C38-C53, C47-C59, C50-C78, C52-C66, and C71-C75. This composition makes it a highly stable molecule.^{1, 3}

Hevein-like peptides, such as WAMP1b, contain a chitin binding domain as a structural motif of 35 amino acids with specific cysteine and glycine residues. Given this, the peptide is able to successfully bind to chitin, acting as a plant defense mechanism against fungi and certain insects.³

WAMP1b also inhibits fungalysin Fv-cmp, a protease produced by Fusarium fungi as a counterattack mechanism to plant’s defenses, that cleaves chitinases. By inhibiting this protease, WAMP1b acts as an additional defense mechanisms against fungal pathogens. It has also been observed that this peptide directly inhibits hyphal elongation.⁴

In an investigation done by Slavokhotova et al., (2014), WAMP1b was tested against various fungi, and it was proved effective against against F. verticillioides with an IC50 of 2.7 µg/ml.

Characterization of Expressible Wheat antimicrobial peptide 1b

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Our DNA sequence (WAMP1b) was synthesized by IDT®️ with the Biobrick prefix and suffix flanking the composite part. This made possible the correct digestion with restriction enzymes EcoRI-HF and PstI. After the digestion, ligation was performed with T7 ligase in order to place our construct into the pSB1C3 linearized backbone with chloramphenicol resistance, which was previously digested with the same restriction enzymes. Using the SnapGene®️ software, we could model our ligated expression plasmid, and the final part resulted in a sequence of 2,590 bp. Thereupon,E. coli SHuffle was transformed by heat shock for following antibiotic selection of clones.

Sequence and Features

References

1.Dubovskii, P. V., Vassilevski, A. A., Slavokhotova, A. A., Odintsova, T. I., Grishin, E. V., Egorov, T. A., & Arseniev, A. S. (2011). Solution structure of a defense peptide from wheat with a 10-cysteine motif. Biochemical and Biophysical Research Communications, 411(1), 14–18. doi: 10.1016/j.bbrc.2011.06.058
2. Istomina, E. A., Slavokhotova, A. A., Korostyleva, T. V., Semina, Y. V., Shcherbakova, L. A., Pukhalskij, V. A., & Odintsova, T. I. (2017). Genes encoding hevein-like antimicrobial peptides WAMPs in the species of the genus Aegilops L. Russian Journal of Genetics, 53(12), 1320–1327. doi: 10.1134/s1022795417120043
3. Odintsova, T. I., Vassilevski, A. A., Slavokhotova, A. A., Musolyamov, A. K., Finkina, E. I., Khadeeva, N. V., … Egorov, T. A. (2009). A novel antifungal hevein-type peptide fromTriticum kiharaeseeds with a unique 10-cysteine motif. FEBS Journal, 276(15), 4266–4275. doi: 10.1111/j.1742-4658.2009.07135.x
4. Slavokhotova, A. A., Naumann, T. A., Price, N. P. J., Rogozhin, E. A., Andreev, Y. A., Vassilevski, A. A., & Odintsova, T. I. (2014). Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases. FEBS Journal, 281(20), 4754–4764. doi: 10.1111/febs.13015