I'm using pCP20 vector carrying gene for FLP recombinase to remove kanamycin resistance cassette from bacteria genome. I know that there are two version of this vector: temperature or L-arabinose inductable (I use the secon one). After following all procedure I didn't obtain kanamycin resistance cassette removing from genome. If anyone use the same vector and could give me some clue I would be very gratetuf. Description of my procedure:
I transformed my bacteria with pCP20, incubate in 30C/180 rpm 2h, then spread them on MSB+Amp plates - they grown about 24h. Then I picked some colonies and put each of them (6 clones) to separated liquid culture (MSB+Amp) and incubated them ON/30C.180rpm. Next day I diluted culture 20x and incubated in 30C/180rpm with 0,2% L-arabinose in MSB+Amp thill they reach OD=1 (I added L-arabinose when OD was 0 and they grown about 6-7h), then I diluted them 20x and incubated them in 42C/220 rpm in MSB without antibiotics. After that, I diluted bacteria 1 mln times and spread them on MSB plate and incubated them ON. Next, I picked 80 colonies and patched them on: Km+MSB, Amp+MSB, MSB plates (in this order) and incubate Km+MSB and MSB at 37C and Amp+MSB at 30C ON. On this step I didn't obtain any colonies in Amp+MSB but all my clones grown in Km+MSB which means that they didn't loose kanamycin resistance cassette. I tried to change L-arabinose concentration to: 0,1 ; 0,5 or 3% but I obtain exactly the same result.
Edyta
Perhaps the host strain you are using carries mutations abolishing arabinose transport? For this experiment you should, ideally, use an AraC+ and AraFGH+ strain. There is a second arabinose transporter (araE) but this one is low affinity and I don't know if it would work for your purposes...
Hope it helps!
Thank you so much for your suggestion! I will check l-ara transport in my strain immediately
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