Biomedical Engineering Reference
In-Depth Information
determinants, and details are presented below for
three of these:
in vivo
expression technology (IVET),
differential fluorescence induction and signature-
tagged mutagenesis.
when it infects an animal or plant host (Mahan
et al.
1993). The basis of the system is a plasmid carrying
a promoterless operon fusion of the
lacYZ
genes
fused to the
purA
or
thyA
genes downstream of a
unique
BglII
cloning site (Fig. 14.11). This operon
fusion was constructed in a suicide-delivery plas-
mid. Cloning of pathogen DNA into the
BglII
site
results in the construction of a pool of transcrip-
tional fusions driven by promoters present in the
In vivo
expression technology (IVET)
IVET was developed to positively select those genes
that are induced specifically in a microorganism
Bgl
II
thyA
lacZ
+
IVET
plasmid
Salmonella
DNA
Cut with
Bgl
II
and ligate
Pl
thyA
lacZ
Transfer to thyA strain of
Salmonella
.
Grow in presence of thymine
Isolate pathogen on
media + thymine + Xgal
White
colonies
Blue
colonies
thyA expressed
from promoter
only active
in vivo
thyA expressed
from promoter
active
in vitro
Fig. 14.11
The basic principle of IVET.
See text for details.
