hygromycin plates, these should be patch tested on
gentamicin.
15. Hyg
R Gm
S transformants can be screened by colony PCR and
Southern Blotting, as described in Subheading 3.2, to confirm
the deletion of the genomic copy of the gene(s) and the introduction of the gene homolog etc.
References
1. Ehrt S, Rhee K, Schnappinger D (2015) Mycobacterial genes essential for the pathogen’s survival in the host. Immunol Rev 264:319–326
2. Stephan J, Stemmer V, Niederweis M (2004)
Consecutive gene deletions in Mycobacterium
smegmatis using the yeast FLP recombinase.
Gene 343:181–190
3. Pavelka MS Jr, Jacobs WR Jr (1999) Comparison of the construction of unmarked deletion
mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Gue ´rin, and
Mycobacterium tuberculosis H37Rv by allelic
exchange. J Bacteriol 181:4780–4789
4. Parish T, Stoker NG (2000) Use of a flexible
cassette method to generate a double
unmarked Mycobacterium tuberculosis tlyA
plcABC mutant by gene replacement. Microbiology 146:1969–1975
5. Knipfer N, Seth A, Shrader TE (1997)
Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise
replacement of the pyrF gene. Plasmid
37:129–140
6. Hinds J, Mahenthiralingam E, Kempsell KE
et al (1999) Enhanced gene replacement in
mycobacteria. Microbiology 145:519–527
7. Triccas JA, Parish T, Britton WJ et al (1998) An
inducible expression system permitting the efficient purification of a recombinant antigen
from Mycobacterium smegmatis. FEMS Microbiol Lett 167:151–156
8. Carroll P, Muttucumaru DGN, Parish T
(2005) Use of a tetracycline-inducible system
for conditional expression in Mycobacterium
tuberculosis and Mycobacterium smegmatis.
Appl Environ Microbiol 71:3077–3084
9. De Smet KAL, Kempsell KE, Gallagher A et al
(1999) Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis.
Microbiology 145:3177–3184
10. Brown AC, Parish T (2006) Instability of the
acetamide-inducible expression vector pJAM2
in Mycobacterium tuberculosis. Plasmid
55:81–86
11. Al-Zarouni M, Dale JW (2002) Expression of
foreign genes in Mycobacterium bovis BCG
strains using different promoters reveals instability of the hsp60 promoter for expression of
foreign genes in Mycobacterium bovis BCG
strains. Tuberculosis 82:283–291
12. Chawla M, Das Gupta SK (1999)
Transposition-induced structural instability of
Escherichia coli-mycobacteria shuttle vectors.
Plasmid 41:135–140
13. Parish T, Lewis J, Stoker NG (2001) Use of the
mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality. Tuberculosis 81:359–364
14. Parish T, Stoker NG (2000) glnE is an essential
gene in Mycobacterium tuberculosis. J Bacteriol
182:5715–5720
15. Parish T, Stoker NG (2002) The common aromatic amino acid biosynthesis pathway is essential
in
Mycobacterium
tuberculosis.
Microbiology 148:3069–3077
16. Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.
FEMS Microbiol Lett 229:211–215
17. Cole ST (1999) Learning from the genome
sequence of Mycobacterium tuberculosis
H37Rv. FEBS Lett 452:7–10
18. Camus J-C, Pryor MJ, Medigue C et al (2002)
Re-annotation of the genome sequence of
Mycobacterium tuberculosis H37Rv. Microbiology 148:2967–2973
19. Strong M, Mallick P, Pellegrini M et al (2003)
Inference of protein function and protein linkages in Mycobacterium tuberculosis based on
prokaryotic genome organization: a combined
computational approach. Genome Biol 4:R59
20. Parish T, Roberts G, Laval F et al (2007) Functional complementation of the essential gene
fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG, but not Escherichia
coli fabG. J Bacteriol 189(10):3721–3728
Gene Switching and Essentiality Testing
299
gentamicin.
15. Hyg
R Gm
S transformants can be screened by colony PCR and
Southern Blotting, as described in Subheading 3.2, to confirm
the deletion of the genomic copy of the gene(s) and the introduction of the gene homolog etc.
References
1. Ehrt S, Rhee K, Schnappinger D (2015) Mycobacterial genes essential for the pathogen’s survival in the host. Immunol Rev 264:319–326
2. Stephan J, Stemmer V, Niederweis M (2004)
Consecutive gene deletions in Mycobacterium
smegmatis using the yeast FLP recombinase.
Gene 343:181–190
3. Pavelka MS Jr, Jacobs WR Jr (1999) Comparison of the construction of unmarked deletion
mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Gue ´rin, and
Mycobacterium tuberculosis H37Rv by allelic
exchange. J Bacteriol 181:4780–4789
4. Parish T, Stoker NG (2000) Use of a flexible
cassette method to generate a double
unmarked Mycobacterium tuberculosis tlyA
plcABC mutant by gene replacement. Microbiology 146:1969–1975
5. Knipfer N, Seth A, Shrader TE (1997)
Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise
replacement of the pyrF gene. Plasmid
37:129–140
6. Hinds J, Mahenthiralingam E, Kempsell KE
et al (1999) Enhanced gene replacement in
mycobacteria. Microbiology 145:519–527
7. Triccas JA, Parish T, Britton WJ et al (1998) An
inducible expression system permitting the efficient purification of a recombinant antigen
from Mycobacterium smegmatis. FEMS Microbiol Lett 167:151–156
8. Carroll P, Muttucumaru DGN, Parish T
(2005) Use of a tetracycline-inducible system
for conditional expression in Mycobacterium
tuberculosis and Mycobacterium smegmatis.
Appl Environ Microbiol 71:3077–3084
9. De Smet KAL, Kempsell KE, Gallagher A et al
(1999) Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis.
Microbiology 145:3177–3184
10. Brown AC, Parish T (2006) Instability of the
acetamide-inducible expression vector pJAM2
in Mycobacterium tuberculosis. Plasmid
55:81–86
11. Al-Zarouni M, Dale JW (2002) Expression of
foreign genes in Mycobacterium bovis BCG
strains using different promoters reveals instability of the hsp60 promoter for expression of
foreign genes in Mycobacterium bovis BCG
strains. Tuberculosis 82:283–291
12. Chawla M, Das Gupta SK (1999)
Transposition-induced structural instability of
Escherichia coli-mycobacteria shuttle vectors.
Plasmid 41:135–140
13. Parish T, Lewis J, Stoker NG (2001) Use of the
mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality. Tuberculosis 81:359–364
14. Parish T, Stoker NG (2000) glnE is an essential
gene in Mycobacterium tuberculosis. J Bacteriol
182:5715–5720
15. Parish T, Stoker NG (2002) The common aromatic amino acid biosynthesis pathway is essential
in
Mycobacterium
tuberculosis.
Microbiology 148:3069–3077
16. Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.
FEMS Microbiol Lett 229:211–215
17. Cole ST (1999) Learning from the genome
sequence of Mycobacterium tuberculosis
H37Rv. FEBS Lett 452:7–10
18. Camus J-C, Pryor MJ, Medigue C et al (2002)
Re-annotation of the genome sequence of
Mycobacterium tuberculosis H37Rv. Microbiology 148:2967–2973
19. Strong M, Mallick P, Pellegrini M et al (2003)
Inference of protein function and protein linkages in Mycobacterium tuberculosis based on
prokaryotic genome organization: a combined
computational approach. Genome Biol 4:R59
20. Parish T, Roberts G, Laval F et al (2007) Functional complementation of the essential gene
fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG, but not Escherichia
coli fabG. J Bacteriol 189(10):3721–3728
Gene Switching and Essentiality Testing
299