Biomedical Engineering Reference
In-Depth Information
1
Construct 10
17
oligonucleotide tags with
the structure
(NK)
20
Environomics
The techniques described above to identify bacterial
virulence genes can be applied to other microbe -
host interactions, e.g. protozoan infections of humans,
bacterial or fungal pathogenesis of plants, or even
beneficial rhizosphere-microbe interactions (Rainey
1999). Essentially, these techniques are methods
for scanning the entire microbial genome for genes
that are expressed under particular environ-
mental conditions. This approach has been termed
'environomics'.
2
Clone tags into transposon
Tn5
resident in plasmid vector.
3
Transform tagged transposon plasmids into
E.coli
selecting
for kanamycin resistance. The individual transformants are
not isolated but kept as a pool.
Theme 4: Protein engineering
4
Conjugate plasmids into
Salmonella
and select for transposition
of tagged transposon. Each kanamycin resistant cell should
carry a different tag.
Introduction to theme 4
One of the most exciting aspects of recombinant
DNA technology is that it permits the design, devel-
opment and isolation of proteins with improved
operating characteristics and even completely novel
proteins. The simplest example of protein engineer-
ing involves site-directed mutagenesis to alter key
residues, as originally shown by Winter and col-
leagues (Winter
et al.
1982, Wilkinson
et al.
1984).
From a detailed knowledge of the enzyme tyrosyl-
tRNA synthetase from
Bacillus stearothermophilus
,
including its crystal structure, they were able to
predict point mutations in the gene that should
increase the enzyme's affinity for the substrate ATP.
These changes were introduced and, in one case, a
single amino acid change improved the affinity for
ATP by a factor of 100. Using a similar approach, the
stability of an enzyme can be increased. Thus Perry
and Wetzel (1984) were able to increase the ther-
mostability of T4 lysozyme by the introduction of a
disulphide bond. However, although new cysteine
residues can be introduced at will, they will not
necessarily lead to increased thermal stability (Wetzel
et al.
1988).
5
Arrange each individual kanamycin resistant clone, which
should be result of different insertion event and will have
different tag, in 8
×
12 grid in 96 well microtitre plates.
6
Pool groups of 96 clones and infect mouse. After period for
infection to be established, re-isolate kanamycin resistant cells
from mouse. Extract DNA.
7
Screen DNA from mouse isolates and determine which tags
are absent relative to those that are present in microtitre trays.
Tags that are missing must be attached to transposons that
have inserted into a gene essential for mouse virulence.
Fig. 14.12
The basic methodology for signature tagged
mutagenesis.
virulence. In this way 28 different mutants with
attenuated virulence were identified and some of
these mutants were in genes not previously identified.
The principle of signature-tagged mutagenesis
has been extended to the analysis of pathogenicity
determinants in a wide range of bacteria (for a review,
see Handfield & Levesque 1999) and to fungi (Brook-
man & Denning 2000).
Improving therapeutic proteins with
single amino acid changes
As noted earlier (p. 283), many recombinant
proteins are now being used therapeutically. With
some of them, protein engineering has been used to
generate second-generation variants with improved
