Construction of biofilm formation related mutants in Vibrio parahaemolyticus

Objective To construct the mutants of biofilm related genes in Vibrio parahaemolyticus and confirm the mutants.Methods The homologous upstream and downstream flanking fragments of target gene were amplified by using PCR,and the fusion homologous fragment was amplified by using the two flanking fragments as template.Then the fusion homologous fragment was digested by restriction enzyme and cloned into suicide plasmid pDS132.The recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD 2210633 through conjugation.The mutants were screened and identified by PCR and the phenotype of one mutant was analyzed in order to verify that the mutants were constructed successfully.Results Six recombinant plasmids carrying the fusion homologous fragments of genes vbfR,crp,hns,swrZ,swrT and cpsR respectively were constructed and identified by PCR.The amplification products of 1190,1128,1136,953,1242 and 1112 bp were obtained respectively.The six mutants (ΔvbfR,Δcrp,Δhns,ΔswrZ,ΔswrT and ΔcpsR) were constructed using recombinant plasmids.Verified by PCR,the size of amplification products of mutants (1190,1128,1136,953,1242 and 1112 bp respectively) was less (610,739,421,542,427 and 1367 bp respectively) than the corresponding positive control.Meanwhile,none of the products was amplified using the primers locating on the target gene.One mutant Δhns was selected to test the ability of biofilm formation.The result showed that the ability of biofilm formation of mutant Δhns was increased compared with the wild type.Conclusion Six mutants of biofilm related genes in Vibrio parahaemolyticus were constructed and tested by molecular and phenotype experiment to confirm that the mutants were constructed successfully. Key words: Vibrio;  Plasmids;  Biofilms;  Gene knockout techniques;  Mutation