You need to provide more information before anyone can answer. What organism are you using? How did you generate the knockouts? Are the genes in an operon or not?

I am using salmonella enterica serovar Typhimurium. Yes genes are in operon. Th e recombination was done using red recombinase kit

Depending upon how you created the mutation, for example if you inserted a drug cassette into a gene to knock it out, you may have blocked expression of downstream genes due to transcriptional polarity.

we have done the whole genome sequencing for knockout and didnot find any problem with the polarity . The knockout for two different genes are giving same kind of phenotype , can it be possible?

Sequencing is not going to tell you about transcriptional polarity. were the knockouts done with an insertion cassette or a deletion? And are they early genes in the operon or distal ones?

The two knockout genes giving the same phenotype, without more details just can’t comment on it. What are the genes and what is the phenotype?

its and insertion cassette..these are dna binding proteins

Polarity is a major concern during gene knock outs. if your gene is part of an operon, then there are more chances of transcriptional/translational polarity. You can test this by RT-qPCR of downstream genes and compare expression with WT.

Second thing is complementing in pBAD vector. We traditionally complement the gene with its native promoter. However, here you are using pBAD promoter which activates only in the presence of arabinose. So your complementing conditions may be a problem. For STM, gene complementation is done in pWSK plasmids. Article Construction of versatile low-copy-number vectors for clonin...