Obtain double gene knockout mutant strains of E. coli from single mutant strains. (Keio collection)
Protocol
From the single mutant strains ΔcyaA, the first step was to remove the Kan cassette in order to create the recipient strain. The second step is using the P1 bacteriophage to transfer a Kan cassette from another bacteria to this recipient strain, in order to replace the second gene. The phage is grown on a strain containing the Kan cassette (ΔaraC or 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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