Gene reconstitution using high efficiency homologous recombination between a bacteriophage fd and a plasmid.
Highly efficient intermolecular crossing-over was observed occurring between regions of limited homology in a fd filamentous phage and a plasmid. These extraneous regions corresponded to two overlapping fragments of the beta-lactamase gene. Gene reconstitution through homologous recombination of these regions yielded a highly ampicillin-resistant phenotype in Escherichia coli while co-expression of the enzyme fragments afforded low and thermosensitive activity. The recombination rates were between two and three orders of magnitude higher than that reported between plasmids using a similar assay. The fd-plasmid cointegrate was detected in recombined bacteria, as was its encapsidation into phage particles and subsequent transduction. A 100-fold reduction in the recombination rate was observed in a recA mutant strain even though crossing-over was still efficient. This gene reconstitution strategy is generally applicable to phage display technology and provides an easy way for constructing large combinatorial libraries of mutants.
Brédo
Anne
A
Laboratoire de Biochimie Physique et des Biopolymères, Institut des Sciences de la Vie, Université catholique de Louvain, Place L. Pasteur, 1, B 1348 Louvain la-Neuve, Belgium.
Mathonet
Pascale
P
Deschuyteneer
Geneviève
G
Soumillion
Patrice
P
D017104:Bacteriophage M13 / Q000235:genetics*; D004926:Escherichia coli / Q000502:physiology*; D015321:Gene Rearrangement / Q000235:genetics*; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins