antibiotic/selection markers used. The first switching vector system
involves the construction of a Δ
0 int strain using pAPA3 [20], which
carries the antigen 85A promoter, gentamicin resistance marker,
mycobacteriophage L5 integrase coding region, and the attP site.
This can then be switched for a hygromycin version called pHAPY1
(Fig. 3), either as an empty vector or containing gene homologs,
truncated gene sequences, etc. The other strategy involves cloning
the gene of interest with its own promoter region into a standard
E. coli cloning vector into which a “Gm-Int” cassette is introduced.
This cassette holds the gentamicin resistance marker, mycobacteriophage L5 integrase, and the attP site and can be introduced into
the cloning vector by a simple HindIII digest and ligation (Fig. 4).
A switching version can be made by the introduction of a “HygInt” cassette, where the resistance marker is hygromycin. Again
these methodologies are based on a SCO strain made using the
method described in [4]; other SCO creation methods would still
work, but are likely require an adaption of the antibiotic/selection
markers used.
Table 1
Vector strategies for switching
Name
Selection markers
Promoter
pAPA3
Amp
R , Gm
R
Ag85A
Unique PacI cloning site—Fig. 1
pHAPY1
Amp
R , Hyg
R
Ag85A
Unique PacI cloning site—Fig. 1
Gm-Int cassette
Gm
R
None
Excisable as a HindIII fragment—Fig. 2
Hyg-Int cassette
Hyg
R
None
Excisable as a HindIII fragment—Fig. 2
Pac I
a
b
Pac I
ß – lactamase
ß – lactamase
HygR
L5 integrase
attP
Gm
attP
L5 Int
pAPA3
(5928 bp)
pHAPY1
(6677 bp)
Fig. 3 Vectors carrying the antigen 85A promoter, suitable for creation of delinquent strains via merodiploid,
pAPA3 (Gm
R
), and switching pHAPY1 (Hyg
R
)
Gene Switching and Essentiality Testing
289