Part:BBa_K4147007

PcOSM Construct with DsbA

This part contains the PcOSM expression construct (BBa_K4147003) with co-expression of DsbA (BBa_K4147006) for disulfide bond formation and proper folding of the osmotin protein.

Usage and Biology

This osmotin is a plant defense protein with a molecular weight of ~24 kDa. PcOSM belongs to pathogenesis related-5 (PR-5) family, which is accumulated in response to both biotic and abiotic stresses [1]. This osmotin has exhibited significant inhibitory activity against the oomycete pathogen Phytophthora capsici. This protein is able to inhibit fungal growth through inhibition of hyphal growth, spore germination, spore lysis, and reduction in viability of spore [2].

This part codes for an osmotin native from Phytophthora resistant wild Piper colubrinum [3]. The codons of this sequence were optimized for expression in E. coli. This gene encodes for a 230 aminoacids peptide fused with a 6XHis Tag. PcOSM have shown significant inhibitory activity against P. capsici, which is one of the most devastating pathogens infecting chilli crops [1]. Also, it has shown inhibitory activity against other fungal pathogens as Fusarium oxysporum [3]. Several experiments suggest that the mechanism of action of this osmotin is membrane permeabilization due to the positive charge of the osmotin [3]. Also, it has been hypothesized that the fungal inhibition shown by osmotin is due to the induction of ROS production [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 [4]. 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 [4].

===Sequence and Features===

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 806
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1184
  Illegal NgoMIV site found at 1368
  Illegal AgeI site found at 498
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization of PcOSM 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.

Figure 1. SnapGene®️ map of BioBrick BBa_K4147007.

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.

Figure 2.Left. SnapGene®️ amplification through PCR of BBa_K4147007 on an 0.8% agarose gel with NEB Quick-Load Purple 1Kb Plus DNA Ladder. Right F) Amplification of PcOSM Construct with LacI regulated promoter where the highlighted band corresponds to approximately 1,797 bp.

REFERENCES

[1] Geetha RG, Krishnankutty Nair Chandrika S, Saraswathy GG, Nair Sivakumari A, Sakuntala M. ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin. Molecules. 2021; 26(8):2239. https://doi.org/10.3390/molecules26082239

[2] Chowdhury, S., Basu, A. & Kundu, S. Cloning, Characterization, and Bacterial Over-Expression of an Osmotin-like Protein Gene from Solanum nigrum L. with Antifungal Activity Against Three Necrotrophic Fungi. Mol Biotechnol 57, 371–381 (2015). https://doi.org/10.1007/s12033-014-9831-4

[3] Mani T, Sivakumar KC, Manjula S. Expression and functional analysis of two osmotin (PR5) isoforms with differential antifungal activity from Piper colubrinum: prediction of structure--function relationship by bioinformatics approach. Molecular biotechnology. 2012;52(3):251–261.

[4] 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