MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The “polar effect” of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of ValS phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph wt from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying λattL-CmR-λattR and transduced with phage P1 into MG1655; later, the CmR marker was removed with the use of λXis/Int recombinase. Parallel to this procedure, a spontaneous ValR mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of L-Val (50 μg/ml) was isolated. It was shown that a nucleotide deletion in the isolated ValR strain had been generated in the region of the identified ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the ValR phenotype in the obtained strain, was transferred to MG1655-rph wt using cotransduction, by analogy to the transfer of rph wt. Evaluation of rates of aerobically growing cells (μ, hour-1) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655,MG1655-rph wt, and MG1655-(rph wt, ilvG-15), respectively.
Russian Journal of Genetics – Springer Journals
Published: Mar 23, 2010
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