Vibrio Flagella Deletion Mutant Construction

Bacterial strains and plasmids used in this study are listed in Table 1. Regarding the deletion mutant of ZSW5 (pomAB) and ZSW6 (pomAB and fliL), 500 bp sequence upstream of the fliL or pomA start codon flanking with a SacI site at its 5′ end and 500 bp sequence downstream of the fliL or pomB stop codon flanking a SacI site at 3′ end were PCR amplified and these DNA fragments were together ligated into a pGEM-T cloning vector (Promega). After SacI digestion of this plasmid, the ΔfliL or ΔpomAB DNA fragment containing about 1000 bp was inserted into the suicide vector pSW7848⁵⁸ . Deletion strain ZSW5 and ZSW6 were constructed by using these suicide plasmids according to the published protocol^{59 (link)}. The plasmids of pZSW7 and pZSW8 were generated using a fast-cloning method^{60 (link)}. The constructed plasmids were introduced into E. coli DH5α, and cultured overnight at 37 °C in LB medium (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl) containing 25 μg/ml chloramphenicol for plasmid collection. Plasmids were isolated by QIAprep Spin Miniprep Kit (Qiagen) and introduced into the Vibrio cell by electroporation with parameters of 1.4 kV and 200 W^{61 (link)}. The sequences of all the plasmid constructs were checked using ABI Prism 3130 genetic analyzer (Applied Biosystems).