Construction of pSETsacB plasmid and evaluation of kdpE gene knockout efficiency with and without markers in Aeromonas dhakensis
In order to construct a novel gene-knockout plasmid,a novel plasmid pSETsacB was constructed based on the plasmids pSET4s and pK18mobsacB by using PCR and cloning techniques.Then pK18mobsacB was used as control,the knockout efficiency of kdpE gene in Aeromonas dhakensis was evaluated via the newly constructed plasmid with or without tetr marker.The results showed that there was no difference in the knockout efficiency between the two plasmids with tetr marker,but the efficiency of scarless deletion by pSETsacB was significantly higher than that of the control plasmid pK18mobsacB.pSETsacB is a thermosensitive shuttle plasmid,and is hence expected to play a role in the genetic mechanism of Gram-positive and Gram-negative bacteria.This study also provides important mutant material for investigating the kdpE gene of A.dhakensis.
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