©Nature Biotechnology
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Construction of E.
coli strains for parallel screening. (A) Plasmid-based strategy for
regulating expression of a target gene that has been deleted from the chromosome
(bent lines). PC, PBAD, promoters of the araC
and araBAD gene clusters, respectively; genX, any bacterial
gene X. The plasmid origin of replication (ori) is denoted by the small
shaded box and represents any plasmid origin (from low copy to high copy
number). (B) Recombination using linear, double-stranded DNA molecules and
l-redgam. In this example, the gene for
nicotinamide adenine dinucleotide A replaces the mutant version on the chromosome.
Blunt-ended lines, linear DNA; large X, l-red-mediated
crossover between regions of homologous DNA. (C) Linear DNA recombination
to replace the defective lprophage. att+
refers to a fragment of E. coli chromosomal DNA containing the bacteriophage
lattachment site, attB. (D) Deletion of
target genes on the chromosome using the lox2–kan
cassette. Colored boxes, target genes (murA, fabI, and metG);
shaded boxes, neighboring genes that encode open reading frames; curvy lines,
homologous DNA shared by the linear DNA substrate and the bacterial chromosome.
P1 and P2 designate promoter elements flanking the
target genes; specific promoters for each target are identified by name.
Illustrations are not to scale. (E) Agarose gel showing PCR products generated
from genomic DNA templates of various E. coli strains constructed
with the bacteriophage recombination systems. Lanes 1 and 12: marker DNA
(0.1 kb and 1 kb ladders, respectively). Lanes 2 and 3: removal of transposon
Tn10 from chromosome of DY329; a multiplex PCR using primers within
tetA and flanking nadA shows that the smaller internal band
(0.95 kb) is lost when the wild-type nadA allele is restored (1.45
kb band, lane 3). The faint 0.85 kb band (lane 3) is a nonspecific PCR product
typical of multiplex PCR. Lanes 4 and 5: removal of the lprophage;
a multiplex PCR done with primers flanking and within the prophage shows
the smaller internal band is lost after the wild-type attB allele
is restored. For the metG deletion, PCR was done with an internal
primer (directed against the kan coding sequence) and two primers
flanking metG. Lanes 6–8: PCR products representing metG,
replacement by lox2–kan cassette, and the
PCR product after removal of the kanamycin marker by P1 Cre, respectively.
Lanes 9–11: corresponding PCR products with primers flanking the fabI
coding sequence. ©Nature Biotechnology |