Table 4 Efficiency of introduction of silent mutations into essential genes in E. coli K-12 BW25113 using pSLTS as a helper plasmid
Target gene | frr | ppa | ||
|---|---|---|---|---|
length of gene (bp) affected amino acid residue | 558 Ser33 | 531 | ||
Position of mutation (bp) | 97-99 | 445-447 Ser149 | 109-111 Ser37 | 343-345 Ser115 |
Mutation | TCT➜AGC | AGC➜TCT | AGC➜TCT | AGC➜TCT |
Efficiency of integration of the mutation cassette | 100% (4/4) | 100% (4/4) | 67% (2/3) | 100% (4/4) |
Fraction of colonies that lost the selection marker after double-strand cleavage of the mutation cassette by I-SceI | 2/7 | 3/6 | 4/10 | 9/20 |
Fraction of colonies that lost the selection marker for which a fragment of the correct size was amplified by colony PCR using primers flanking the gene of interest | 2/2 | 3/3 | 2/2 | 4/4 |
Fraction of colonies for which the correct PCR product was obtained that contained the desired mutation | 2/2 | 3/3 | 2/2 | 4/4 |