I am trying to purify a protein out of a BL21 E. coli. I have inserted a pET24b plasmid with the gene into the BL21. The problem is that I can't separate it from an essential homolog in the bacterial genome. My POI is able to rescue cells devoid of this essential homolog. I want to create a knockout of the essential protein in the genome so that when I purify my POI, it has none of the other protein. Can I do this with Lambda Red recombineering? Once I make the knockout, will I be able to add my plasmid in? Or can I do the recombineering with my plasmid at the same time? Will an E. coli take up two plasmids at the same time? My pET has kan resistance, I was thinking about putting the AMP resistance in the middle of my knockout gene, and then theoretically any cell that grows on and AMP/kan plate should have both the knockout and my plasmid, correct?
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