Part:BBa_K2959007

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 insects³.

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

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,Escherichia coli SHuffle was transformed by heat shock for following antibiotic selection of clones.

Figure 1. SnapGene®️ map of BioBrick BBa_K2959007

The next step was to amplify our BioBrick sequence through colony PCR performed upon our transformed cells to confirm the presence of our expression plasmid inside of our chassis. With the help of the specific forward Biobrick prefix [BBa_G1004] and the specific reverse Biobrick suffix [BBa_G1005], we were able to amplify our sequence exclusively. Through an agarose gel we confirmed the correct transformation. The PCR action from SnapGene®️ was used to predict the size of the amplified sequence which resulted in a size of 579 bp.

Figure 2. (On the left)SnapGene®️ amplification through PCR of BBa_K2959007. (On the right) Agarose gel electrophoresis of BBa_K2959007 compared with NEB Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 579 bp.

Protein production

IPTG Induction and Extraction

Following the construction of the BioBrick, it was necessary to induce protein production. Production of WAMP1b was possible under Lacl promoter when induced with 0.2 or 0.4 mM IPTG at 30°C and 225 rpm. This was followed by protein extraction by lysis solution to which lysozyme was added in order to obtain our soluble peptides.

Antifungal Assay

In order to test the antifungal activity of our peptides as well as prove their viability as a mechanism to inhibit Verticillium dahliae, an antifungal susceptibility test on a 96 well plate was carried out by measuring absorbance at 405 nm, wavelength used in standardized protocols to measure growth of filamentous fungi⁵. Due to a lack of time, we couldn’t reach the experimental stage of the project of peptide purification, so experiments were made using soluble protein extracts from our transformed cells’ lysates. Different dilutions of the extracts were prepared which were applied to a spore suspension of V. dahliae. Dilutions of the extracts of untransformed cells were used as controls to prove that inhibition was the result of the peptides and not any other protein contained within the extract. Soluble proteins from E. coli SHuffle were used as control for the WAMP1b extract. Concentrations of extracts with peptides were equalized to their controls. Table 1 details the concentrations of each dilution.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 261
  

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
5. Schwalbe, R., Steele-Moore, L., & Goodwin, A. C. (2007). Antimicrobial susceptibility testing protocols. Crc Press.