Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains

Abstract

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10(12)-10(13) mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.

Figure 1

Schematic representation of reporter-containing plasmids. The reporter genes (big arrows) at close, mid and far positions and the spectinomycin-resistant determinant (thin line) are indicated. The ColE1-type origin is represented by a thin arrow (ori). Replication is initiated by Pol I at the tip of the arrow and progresses clockwise. The areas replicated by Pol I and Pol III are indicated by black and grey lines respectively, the region of Pol I/Pol III transition being arbitrarily localized.

Figure 2

Prolonged growth and mutant frequency. The plasmid containing lacI at the close position was propagated in GMEc6 (Pol I* MutHLS^–) for 150 generations using serial dilutions to obtained successive cycles of growth, and was analyzed for mutation frequency. The relative frequency of lacI mutants is given as a function of the generation number. Results of two independent experiments are considered.

Figure 3

Mutation distribution downstream of the plasmid origin. Twenty LacI^– and 24 Lipase^– independent mutants isolated from GMEc6 (Pol I* MutHLS^–) were sequenced. The thin line represents plasmid sequences, the bent arrow, the initiation site of DNA replication at ori and the bold lines, the reporter genes. Dots denote positions of mutations. In lacI, 14 different mutated bases were observed, one occurred seven times and another twice.