Part:BBa_K4147008
PelB-(CBD)2-DrsB1 Construct with DsbA
This part contains the (CBD)2-DrsB1 expression construct (BBa_K4147001) that its composed by the folloing: a linear construct BBa_K4147001 for (CBD)2-DrsB1 which already incorporates a PelB signal peptide on the N- terminal as well as a 6X-His tag at the end of the dermaseptin CDS (C- terminal) for later protein purification. This composite construct includes a LacI regulated promoter. Additionally, it contains an RBS under the part name BBa_B0032. Also it incorporates the co-expression of DsbA (BBa_K4147006) for disulfide bond formation and proper folding of the dermaseptin protein. Finally, it has a double terminator (BBa_B0015) to ensure correct termination.
Usage and Biology
Among a wide range of AMPs, polycationic dermaseptin peptides are produced by the skin glands of the frog Phyllomedusa bicolor. Although most dermaseptins are known to disrupt pathogens' lipid membranes, which leads to the development of membrane diseases, some may breach the membrane and interfere with critical cell components like DNA [1]. This system makes this AMPs the alternative antibiotics to boost plant innate immune system. It supplies both antibacterial and antifungal protection to a broad range of plant pathogens but shows no toxic effects on plant and mammalian cells [2]. This peptide of molecular weight of 22.1 kDa contains a tandem repeat of a chitin binding domain (CBD) as an improvement of dermaseptin-b1 gene (AKA MsrA2)(BBa_K2577001) which binds to chitin fungal cell wall, thereby increasing the lytic activity of the catalytic domain.
This fusion of DrsB1 to CBD not only protect DrsB1 from host degradation but its affinity for fungal cell wall chitin remains the same. It has been shown showed that this recombinant peptide possessed strong antifungal activity in vitro, inhibiting the growth of mycelia as well as the germination of conidia and spores[1]. This part has been used in the generation of transgenic lines of Tobacco plants which were resistant to Alternaria, Phytophthora, Fusarium, Pythium fungi [2].
The disulfide bonds are essential for structure stabilization and biological functions of these peptides [1]. To increase the solubility and functionality of the protein in E. coli, the N-terminus of the protein is usually fused to a signal peptide such as the DsbA protein. This can cause the heterologous protein to be exported from the cytoplasm into the periplasm or the culture media via the type II secretion system [3]. DsbA was frequently used in the development of commercial expression plasmids to support periplasmic or extracellular secretion based on this property. Adding proteins to DsbA peptides can improve the target molecule's performance and solubility while also drastically reducing the creation of inclusion bodies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1184
Illegal NgoMIV site found at 1368
Illegal AgeI site found at 392
Illegal AgeI site found at 671 - 1000COMPATIBLE WITH RFC[1000]
Characterization of PelB-(CBD)2-DrsB1 Construct with DsbA
Before moving on to the experimental process, we first performed an in silico analysis in SnapGene®️ to simulate our ligated expression plasmid. The theoretical result was a sequence of 3,810 bp, as shown in Figure 1.
Our insert of PcOSM Construct with DsbA was later synthetized by Twist Bioscience with the Biobrick prefix and suffix as well as adapters flanking the composite part for easiest restriction digest. EcoRI and PstI enzymes were used to digest both construct and vector. Once digested we proceed to ligate our insert into a pSB1C3 plasmid with Anza™ T4 DNA Ligase Master Mix. The ligation product was then transformed into E. coli BL21(DE3) cells by heat shock.
The next step was to confirm the presence of the vector of interest in our chassis after transformation, so we performed colony PCR using Forward: 5'-GTTTCTTCGAATTCGCGGCCGCTTCTA and Reverse: 5'-GTTTCTTCCTTCCTGCAGCGGCCGCTACTAG primers specific for the BioBrick prefix and suffix respectively. The PCR action from SnapGene®️ was used to predict the resultant agarose gel.
2-DrsB1.png)
REFERENCES
[1] Shams, M. V., Nazarian-Firouzabadi, F., Ismaili, A., & Shirzadian-Khorramabad, R. (2019). Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity. Molecular Biotechnology. doi:10.1007/s12033-019-00153-x
[2] Khademi, M., Varasteh-Shams, M., Nazarian-Firouzabadi, F., & Ismaili, A. (2020). New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants. Frontiers in Plant Science, 11. doi:10.3389/fpls.2020.01236
[3] Zhang, W., Lu, J., Zhang, S., Liu, L., Pang, X., & Lv, J. (2018). Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microbial Cell Factories, 17(1). doi:10.1186/s12934-018-0894-y
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