Phage-resistant microorganisms

A method for generating genetically modified microorganisms from Escherichia coli (E. coli) strains resistant to infection by phages, the method comprising:

• a) making mutations in tfaD and yejO genes of an E. coli strain that is sensitive to infection by phages;
• b) growing the E. coli strain having mutations in the tfaD and yejO genes in a culture medium, thereby forming a culture of the E. coli strain, until the E. coli strain reaches an optical density at 600 nm from 2 to 5;
• c) adding phages λ, φ80 and T4 to the culture medium, the phages having a concentration greater than 100 plaque-forming units;
• d) contacting the culture of the E. coli strain with the phages for 30 minutes to 6 hours at a temperature of 20° C. to 25° C. to allow for infection by the phages;
• e) allowing for cellular lysis for 1 hour to 8 hours at a temperature of 35° C. to 39° C.;
• f) allowing the culture of the E. coli strain to recover for 1 hour to 24 hours;
• g) isolating colonies into plates with culture medium;
• and
• h) verifying the resistance to infection by phages λ, φ80 and T4 of the E. coli strain generated by steps a) to g) by repeating steps a) to g);
• wherein the adding comprises separately adding the phages λ, φ80 and T4 to the culture medium to form a mixture of phages or adding a mixture containing the phages 2, $80 and T4 to the culture medium,
• wherein a genetically modified microorganism is generated from the E. coli strain resistant to infection by phages λ, Φ80 and T4,
• the method further comprising verifying the resistance to infection by phages λ, φ80 and T4 of the E. coli strain was caused by mutations in the tfaD and yejO genes of said E. coli strain by:
• growing the E. coli strain having mutations in the tfaD and yejO genes in the culture medium in a reactor at different operating conditions of the reactor, the different operating conditions of the reactor comprise one or more of pH, temperature, and/or dissolved oxygen;
• determining the culture of the E. coli strain did not clarify during fermentation;
• and
• sequencing the E. coli strain to corroborate that the mutations in the tfaD and yejO genes of the E. coli strain were the cause of the resistance to infection by the phages λ, φ80 and T4,
• the method also further comprising identifying that mutation in the tfaD gene of the E. coli strain corresponds to a mutated TfaD protein, the mutated TfaD protein containing a mutation at position 69 of said TfaD protein;
• and
• identifying that mutation in the yejO gene of the E. coli strain corresponds to a mutated YejO protein, the mutated YejO protein containing a mutation at position 23 of said YejO protein.