I tried using Normal LB and YPD still no success. I am conjugation using E.coli S17.1.
I successfully used DNA medium for S. venezuelae. Also try to reduce nalidixic acid concentration. Different streptomycetes respond differently to Nal. I would make a titration of different concentrations and see what's the minimal nal that stops E. coli growth but allows your strpetomyces strain to grow. Mg2+ concentration also plays a role in conjugation. Mg2+ affects chromosome structure as far as I know.
Try AS1 agar or modified R2 bottom agar. See media and growth conditions under Methods section of the following article.
PMID: 8828204
DOI: 10.1099/00221287-142-9-2363
Microbiology (1996, 142, 2363-2373)
Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production
Margaret A. McHenney and Richard H. Baltz
I guess that I didn't know the term "DNA media". We grew Streptomycetes on R2 media in order to allow regeneration of protoplasts. Could you define DNA media for me? I haven't worked with Streptomycetes in many years. I am currently studying veterinary nursing through Purdue University, so that is what I am learning about.
You've tried both of those?
Why do you need to generate protoplast? If you are making gene knockouts, there is a much better and easier way called PCR redirect. You can google it and download the manual. Then ask people in John Innes Centre to send you the required engineered E. coli strains. Let me know if you need help
I am an old timer. Protoplasts were once used in transformation of Streptomyces.
Yes, I know but things have changed a lot since :)
It's much easier now. I can send you the required tools if gene KO is what you are after...
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