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I have a few questions regarding molecular genetic techniques in bacterial systems. I've been given a theoretical operon, which I need to knockout/silence in some manner - it consists of an upstream promoter and 5 genes, and covers ~13Kb of the genome. I was wondering what would be the most viable approach to silencing this and to verify the silencing has been made? We've been given a plasmid to use - with a heat sensitive oriC (stable at 30 celsius, not at 42 celsius) which also carries the erythromycin-R gene. Could I use this plasmid for homologous recombination? i.e. insert a random fragment into the middle of the gene. How would I go about constructing such an insert into the plasmid?
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