fulltext.study @t Gmail

Gene reconstitution using high efficiency homologous recombination between a bacteriophage fd and a plasmid

Paper ID Volume ID Publish Year Pages File Format Full-Text
25680 43590 2006 5 PDF Available
Title
Gene reconstitution using high efficiency homologous recombination between a bacteriophage fd and a plasmid
Abstract

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 β-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.

Keywords
Split β-lactamase; Phage display; Homologous recombination; Mutant libraries; RecA
First Page Preview
Gene reconstitution using high efficiency homologous recombination between a bacteriophage fd and a plasmid
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 126, Issue 3, 10 November 2006, Pages 286–290
Authors
, , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering