Bacterial Operon Knockout - Technique Help - (May/04/2012 )

Good Evening,

I'm currently an undergraduate student preparing for a summer research project. I was wondering what the best approach would be to silence the expression of a bacterial operon. I am investigation the function of a 5-gene operon of currently unknown function, with a single identified upstream promoter, thus I need some form of selection with the knockout as the phenotype is unknown.

I was thinking about amplifying a fragment containing the promoter region via PCR and cloning this into an appropriate vector. Mutating the promoter or introducing a random fragment into the promoter and re-introducing this back into the genome via homologous recombination. Though, I am not sure how to go about this or how I would insert such a fragment into the promoter. I could then select for the recombination via PCR or a selectable marker within the fragment (though i'm not sure if anything would be expressed without a dedicated promoter so I think PCR selection is probably more useful)

Many thanks for any help you can offer!

- TJC

Operon means, how long the operon is? We usually follow the paper "
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products" it works well.

christy on Sat May 5 00:35:59 2012 said:

Thank you, I will take a look over this paper. The operon contains five genes, and is ~12Kbp in length. Are there any other approaches?