Obtain double gene knockout mutant strains of E. coli from single mutant strains. (Keio collection)
Protocol
From the single mutant strains, the first step was to remove the Kan cassette in order to create the receiver strain. The second step is using the P1 bacteriophage to transfer a Kan cassette from another bacteria to this receiver strain, in order to replace the second gene. The phage is grown on the strain containing the Kan cassette (ΔaraC and ΔenvZ), and the resulting phage lysate is used to infect the recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome.
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