updated (2014.07.24)
I m constructing a DNA cassette (with genes sacB, SmR, and LacZ), which will replace the galK region of an E. coli strain lacking LacZ after recombination.
1) Anyone has a idea of how reliable works the galK selection?
Also, I am considering other genes for counterselection as sacB sensitivity seems to varies somehow. In previous version of this message, I precised that I will perform scarless recombination using ccdB+cat gene. using a temperature-sensitive plasmid (as pKD46).
Hi, I don't really have experience in your subject but I have a question: to get counterselection, shouldn't the ccdB and ccdA be in different places? From what I understand in your design you have both genes into the Red vector, which is lost after temperature shift?
I'm stating the obvious, but remember to propagate ccdB-containing plasmids in a ccdB-resistant strain (like DB3.1)! (I would not say this if I hadn't happened to me, haha... for at least a few minutes you wonder why your coli has not grown!).
Greetings, thank you I added more precision in the question I modified, yes I would need to propagate ccdB-containing plasmids in a ccdB-resistant strain. The generation of E. coli with CcdB resistance can be apparently easily done (looking at Wang et al. 2013) with the use of 2 complementary oligo primers containing the 2 gyrase-A mutations. Primers are electroporated in E. coli, incubated and mutants selected in L-Arabinose medium.
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