The lambda-red recombinase gene knockout system works very efficiently in Salmonella and has been successfully utilised by researchers across the globe, evidenced by a large volume of publications. The TargeTron system hasnt been used so extensively and if I remember correctly inserts group II introns into a gene of choice. The other advantage of the lambda-red system is that you can remove your inserted antibiotic marker by a second recombination step producing a "cleaner" deletion mutant which has minimal downstream pleiotropic effects. This is particularly important if you are looking at the roles of individual genes within operons.

Thank Anjam and Brian, it is really helpful. But now I have another question: if use the lambda-red method, should I first transform a plasmid with gene coding for recombination protein into Salmonella ?

Hi Yuping, Yes, the recombinase approach requires expression of the phage λ Red recombinase in Salmonella, for efficient recombination between the targeted gene and a linear PCR product. The antibiotic resistance gene can also be excised by the FLP recombinase which acts on the FRT sites ( Datsenko and Wanner, 2000). Expression of the λ Red recombinase or FLP recombinase is required from helper plasmids such as pKD46 and pCP20, respectively. These plasmid are temperature-sensitive so can be removed simply. The pKD46 helper plasmid carries an ampicillin resistance marker (GenBank accession number AY048746), so your Salmonella strain has to be sensitive to this antibiotic.

Hi Yuping,

As mentioned, the Lambda RED system works efficiently in Salmonella for mutagenesis. There are several other methods for mutant construction and one of the 'oldies but goodies' is the SacB system. This is used extensively for generating mutations in a variety of bacterial systems including Salmonella. This process uses the sacB gene, which when expressed in the presence of excess sucrose is lethal, to drive the recombination event. If you have all of the needed tools to use the methods described above, then I would try them first. If you are interested in the SacB method, post back and I can provide you with more detailed protocol/references.

Good luck

There is some serotype variation in how well techniques work, Typhimurium and Enteritidis are amenable to the use of Lambda Red-i most hands it works well. Some others are trickier-the avian adapted S. Pullorum is not very amenable to Lambda Red for example.

Paul Wigley another avian-adapted serotype is Infantis, have you considered any other approach that has worked well? like cas9?