Genetic Engineering of Lactiplantibacillus plantarum for production the nisA as a natural antibacterial protein

Abstract

Currently, researchers are exploring the capability of lactic acid bacteria to produce therapeutic molecules through the use of living microorganisms. This study aims to clone the nisA gene in Lactobacillus plantarium. The process involves isolating the nisA gene from Lactococcus lactis and transferring it to a pET-21a(+) plasmid. To achieve this, PCR fragment carrying the nisA gene and plasmid were predigested by BamHI and HindIII restriction enzymes in appropriate reaction enzyme buffer. To increase the number of plasmids, they were cloned in E. coli DH5α.  Afterward, electroporation was used to transfer the plasmid into L. plantarium. Following this, the transformants were grown in a culture medium that contained ampicillin. Only those bacteria with the pET-21a(+) plasmid were able to grow due to their possession of the ampicillin resistance gene. Sequencing was performed on the obtained clones, confirming the presence of the desired gene. PCR reaction was used to confirm the cloning, and the study demonstrated that nisin protein can be expressed functionally in L. plantarium, increasing its antimicrobial activity. Finally, to enhance the antimicrobial activity of L. plantarum, we performed the transfer of the nisA gene from L. lactis to L. plantarum for the first time by cloning and expression.