A Preliminary Study Of Anabaena Sp. PCC7120 Toxin-antitoxin Gene All7155/asl7156

The toxin and antitoxin system was firstly discovered in the Escherichia coli. Most of them existed in the chromosome of the bacteria, a few of them existed in the low copy plasmid. Through the deeply study of the interaction mechanism of them, the toxin-antitoxin system always existed in the forms of pair:one is toxin gene, another is antitoxin gene. The toxin gene will kill the host bacteria by the mechanism of PSK when the outside environment is not suitable for the growth of the host bacteria. Somtimes the programmed death of the host bacteria will happened or the toxin protein will be translated which will also make the host bacteria dead when the toxin factor was activated.The subject of our study is the gene pair all7155/asl7156 which exist in the alpha plasmid of the Anabaena sp.PCC7120. Their description in TADB is homology of the parD/E toxin-antitoxin system. According to the BLAST of the gene pair and the protein in the NCBI, and the prediction of the secondary structure of the proteins, the gene pair all7155/asl7156 was preliminary sure to be homologous gene of the parD/E toxin-antitoxin system. Based on the analysis of the bioinformatics, use the technique of molecular biology experiments, make the design of primers of the genes, added the special restriction enzyme cutting site. With the pMD18-T as a cloning vector, use the blue-white screening to select the positive monoclonal, and to make sure there is no deletions or mutants by sequencing check. With the pET-30a(+) as a expression vector, construct the recombinant expression plasmid pET-30a-7155 and pET-30a-7156. Using IPTG inducing the expression of the target genes in the E.coli, and optimized the conditions for the expression. The result indicated that the best conditions for inducing the genes expression is IPTG concentration 0.8mM/L, 8 h. Because of the HIS-tag in the expression vector, the study of Western-blot and NI column purification can be made, and the result proved that the NI column was effective to the purification of the HIS-tag fusion protein.At recently research of the toxin-antitoxin system, most of them were type II toxin-antitoxin system, especially the mazE/F family. There were only a few study on the other toxin-antitoxin system, such as the toxin-antitoxin system in the plasmid of the host bacteria. Our study of the gene all7155/asl7156 make some sense, and make the research of parD/E or the toxin-antitoxin system in the plasmid more completely.