I'm planning to integrate my plasmid (containing few gene, an enzyme, antibiotic resistance gene, GFP, lac operon, and others) into E coli expression strain. is there any full protocol for this? thankyou
Here's a nice overview of lambda red system for inserting a cassette into E. coli
https://blog.addgene.org/lambda-red-a-homologous-recombination-based-technique-for-genetic-engineering
John Hardy Lockhart Hi John, thankyou for the reference. is it possible to do this in Rosetta 2 (DE3)pLysS cell? since this strain didn't have lambda red
Based on that blog post, it should be possible to use this system by transforming in the lambda red plasmids prior to recombineering your cassette. After recombination you can remove the lambda red plasmids by incubating the bacteria at 42 ˚C since the lambda red plasmid origin or replication is heat-sensitive.
As a disclaimer, I've never done recombineering on prokaryotes, but this sounds fairly straightforward.
You can introduce the lambda red plasmid into any strain, but note that is for integrating a PCR amplifed cassette into the chromosome by recombination. This is what most people do. However if you really want to integrate a plasmid into the chromosome then you need to use a specific plasmid that has conditional replication so you can select for its integration without replication.
However from what you describe, why do you think you need to integrate your plasmid? Is there some reason you can't just work with a replicating plasmid?
John Hardy Lockhart thank you for the discussion John, really appreciate it. i will give an update about this in here
Michael J. Benedik Hi Michael, thank you for the answer. i believe the main reason my supervisor want me to integrate the gene is to prevent plasmid loss since the 'enzyme' is planned to be used commercially.
i think most commercially produced enzymes are still made from plasmids. But if You wish to integrate I think recombining in your cassette will be preferable over integrating a plasmid
Hello,
maybe this can help you?
Fast and antibiotic free genome integration into Escherichia coli chromosome | Scientific Reports (nature.com)
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