2 Materials
2.1 Construction of a
Complementing Vector
Depending on the strategy you are adopting you should follow
either Subheading 2.1.1—for the creation of a vector where expression is driven by the Ag85a promoter; or Subheading 2.1.2—where
expression is driven by the native promoter.
2.1.1 Construction
of Ag85a Promoter-Driven
Complementing Vector
Using pAPA3
1. pAPA3 (Addgene).
2. PacI.
3. Mycobacterial genomic DNA.
4. Primers designed to amplify the gene to be deleted.
5. PCR reagents, thermocycler, and consumables.
6. PCR cleanup or gel extraction Kit, e.g., QiaQuick, Qiagen, or
similar.
7. Agarose gel electrophoresis equipment.
8. T-A cloning vector, pSC-A (Agilent), or similar.
9. Competent E. coli strains suitable for recombinant genetic
work, e.g., DH5α or XL1.
10. Plasmid purification kit (Qiagen or similar).
11. 50 mg/mL Ampicillin (250Â): Make stock in distilled water,
filter sterilizes, and store in aliquots at À20
C. Use at 200μg/
mL.
12. 50 mg/mL 5-bromo-4-chloro-3-indolyl-B-D-galatopyranoside (X-gal) 1000Â: make stock solution in dimethyl sulphoxide (DMSO) and store at À20
C. Use at a working
concentration of 50μg/mL.
HindIII (5678)
HindIII (3008)
HindIII (3757)
HindIII (6395)
a
b
amp
amp
HygR
L5 integrase
Gm
attP
attP
L5 Int
pUC-Gm-Int
(5678 bp)
pUC-Hyg-Int
(6395 bp)
Fig. 4 Vectors with HindIII excisable L5 integrase cassettes which can be added to a cloning vector, pUC-GmInt (Gm
R
), and pUC-Hyg-Int (Hyg
R
)
290
Amanda Claire Brown
2.1 Construction of a
Complementing Vector
Depending on the strategy you are adopting you should follow
either Subheading 2.1.1—for the creation of a vector where expression is driven by the Ag85a promoter; or Subheading 2.1.2—where
expression is driven by the native promoter.
2.1.1 Construction
of Ag85a Promoter-Driven
Complementing Vector
Using pAPA3
1. pAPA3 (Addgene).
2. PacI.
3. Mycobacterial genomic DNA.
4. Primers designed to amplify the gene to be deleted.
5. PCR reagents, thermocycler, and consumables.
6. PCR cleanup or gel extraction Kit, e.g., QiaQuick, Qiagen, or
similar.
7. Agarose gel electrophoresis equipment.
8. T-A cloning vector, pSC-A (Agilent), or similar.
9. Competent E. coli strains suitable for recombinant genetic
work, e.g., DH5α or XL1.
10. Plasmid purification kit (Qiagen or similar).
11. 50 mg/mL Ampicillin (250Â): Make stock in distilled water,
filter sterilizes, and store in aliquots at À20
C. Use at 200μg/
mL.
12. 50 mg/mL 5-bromo-4-chloro-3-indolyl-B-D-galatopyranoside (X-gal) 1000Â: make stock solution in dimethyl sulphoxide (DMSO) and store at À20
C. Use at a working
concentration of 50μg/mL.
HindIII (5678)
HindIII (3008)
HindIII (3757)
HindIII (6395)
a
b
amp
amp
HygR
L5 integrase
Gm
attP
attP
L5 Int
pUC-Gm-Int
(5678 bp)
pUC-Hyg-Int
(6395 bp)
Fig. 4 Vectors with HindIII excisable L5 integrase cassettes which can be added to a cloning vector, pUC-GmInt (Gm
R
), and pUC-Hyg-Int (Hyg
R
)
290
Amanda Claire Brown