I have been unsuccessful in knocking out bcsQ in Pectobacterium carotovorum ssp. brasiliense using a three-step PCR method.

Plasmids used: pKD4 and pKD46

bcsQ is MinD homologue part of the cellulose synthase operon.

The upstream homologous arm of the PCR cassette is 372bp, the kanamycin containing fragment is 1.5kb and the downstream arm is 480bp in length. 

I successfully generate the kanamycin-containing cassette and obtain transformants.

Screening of transformants (colony PCR) indicates that the cassette has integrated elsewhere in the genome and bcsQ has not been deleted.

I have repeated the transformation with a newly generated cassette and obtain the same results.

I would appreciate any thoughts/suggestions regarding this matter

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