Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains.
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.
Fabret
C
C
Unité de Génétique Microbienne, Domaine de Vilvert INRA, 78352 Jouy-en-Josas, France.
Poncet
S
S
Danielsen
S
S
Borchert
T V
TV
Ehrlich
S D
SD
Jannière
L
L
D001426:Bacterial Proteins / Q000235:genetics; D015169:Colony Count, Microbial; D004252:DNA Mutational Analysis; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D029968:Escherichia coli Proteins; D005787:Gene Frequency / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D056968:Lac Repressors; D008049:Lipase / Q000235:genetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D012641:Selection, Genetic; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D008049:Lipase