Toxin-antitoxin systems (TAS) are common genetic elements in prokaryotic organisms. They consist of a toxin-encoding gene, which in its expressed protein form inhibits bacterial growth, and an antitoxin, which neutralizes the toxin. Based on bioinformatic analyses, TAS are also present in the genome of the cyanobacterium Microcystis aeruginosa PCC 7806, an organism ecologically recognized as a contributor to freshwater blooms and, under certain conditions, a producer of the toxin microcystin, which poses a threat to humans and animals.
Despite the ecological significance of M. aeruginosa, its TAS remain poorly studied, with the majority remaining unconfirmed experimentally. In this work, we aimed to molecularly clone and express three toxin-antitoxin (TA) gene pairs in Escherichia coli. Based on a bioinformatic analysis, we selected the following candidates: BH695_3840 (toxin) - BH695_3841 (antitoxin), WP_036399356.1 (toxin) - BH695_4980 (antitoxin), and WP_002748554.1 (toxin) - WP_036402927.1 (antitoxin). Using genomic DNA, we amplified the selected TA gene pairs via PCR, isolated them, and inserted them into cloning vectors, which were subsequently used to transform E. coli. The transformed bacteria were propagated, and cloning vectors were isolated and subjected to restriction endonuclease digestion to obtain the TA gene pairs. The resulting cloning regions were then inserted into expression vectors. Finally, test expression of the constructs was performed in transformed E. coli cells. Only the BH695_3841 protein was successfully expressed, due to an error in the design of the initial nucleotides for the other constructs.
|
