Difference between revisions of "Part:BBa K4147003"

Latest revision as of 11:37, 12 October 2022

PcOSM Construct with LacI regulated promoter

This part contains the linear construct for pcOSM (plant osmotin from Piper colubrinum) with an included LacI promoter. Additionally, it contains an RBS under the part name BBa_B0032, as well as a 6X-His tag at the end of the osmotin CDS for later protein purification. Finally, it has a double terminator (BBa_B0015) to ensure correct termination. As a linear construct, it contains the RFC10 prefix and suffix, making the part compatible with other iGEM parts.

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].

Sequence and Features

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 3,283 bp, as shown in Figure 1.

Our insert of PcOSM Construct with LacI regulated promoter 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 pTwist Kan High Copy 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.

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.