5. pSC-A (Agilent) is a very useful vector for this purpose and it
has been shown to give a high efficiency when cloning mycobacterial DNA. It also has a unique HindIII site which allows
for the introduction of the Gm-int or Hyg-int cassette.
6. If creating a switching version, use pHAPY1 (HygR).
7. Although pAPA3 has a gentamicin marker, it also holds Amp
R
,
which works better with E. coli than gentamicin for the obtaining of transformants. Transformants can be patch tested onto
LA-Gm when obtained.
8. The Gm-Int cassette (3.0 kb) and pUC vector backbone are of
similar size, therefore the use of 0.6% agarose gels allows for
better separation. The Hyg-Int cassette is easier to separate
(3.7 kb).
9. The efficiency of obtaining E. coli transformants directly on
LA-Gm is very low. Therefore, provided that the T-A cloning
vector was Amp
R
, plate the transformants onto LA-Amp, and
then patch transformants onto LA-Gm.
10. Orientation of the insert and the Gm-int cassette is not critical,
but it can be useful to know for when performing Southern
analysis later on.
11. For Southern analysis, DNA extracts can be made as described
in Subheading 3.2, step 21 of this chapter, and then cleaned
further as described in Chapter 20 “Whole genome sequencing
of Mycobacterium tuberculosis directly from sputum samples”,
of this volume, Subheading 3.1, step 10 onwards.
12. Delinquents have only the integrated gene copy. Proof of
essentiality is established if no DCOs are obtained in the wildtype background but can be obtained when a functional copy
of the gene is introduced. Further confirmation of essentiality
is made when switching for an empty vector shows that the
integrated copy is required for cell viability, e.g., it cannot be
removed.
13. Typically we use pUC-Hyg-Int for switching individual complementing vectors out of the chromosome. If testing which
genes are essential in an operon, or different gene homologs,
pHAPY1 can be engineered to contain these.
14. In the case of essential genes, there should be no transformants
on the hygromycin plates, and a lawn of growth on the gentamicin. Sometimes the vectors cointegrate into the chromosome, resulting intransformants possessing both Hyg
R and
Gm
R . In this case, typically the numbers of colonies on the
gentamicin plus hygromycin plates and hygromycin only plates
will be similar; if colonies are patched onto gentamicin, they
will be resistant indicating that switching has not occurred. For
nonessential genes, or where the gene being switched in is a
functional homolog, transformants should be obtained on the
298
Amanda Claire Brown
has been shown to give a high efficiency when cloning mycobacterial DNA. It also has a unique HindIII site which allows
for the introduction of the Gm-int or Hyg-int cassette.
6. If creating a switching version, use pHAPY1 (HygR).
7. Although pAPA3 has a gentamicin marker, it also holds Amp
R
,
which works better with E. coli than gentamicin for the obtaining of transformants. Transformants can be patch tested onto
LA-Gm when obtained.
8. The Gm-Int cassette (3.0 kb) and pUC vector backbone are of
similar size, therefore the use of 0.6% agarose gels allows for
better separation. The Hyg-Int cassette is easier to separate
(3.7 kb).
9. The efficiency of obtaining E. coli transformants directly on
LA-Gm is very low. Therefore, provided that the T-A cloning
vector was Amp
R
, plate the transformants onto LA-Amp, and
then patch transformants onto LA-Gm.
10. Orientation of the insert and the Gm-int cassette is not critical,
but it can be useful to know for when performing Southern
analysis later on.
11. For Southern analysis, DNA extracts can be made as described
in Subheading 3.2, step 21 of this chapter, and then cleaned
further as described in Chapter 20 “Whole genome sequencing
of Mycobacterium tuberculosis directly from sputum samples”,
of this volume, Subheading 3.1, step 10 onwards.
12. Delinquents have only the integrated gene copy. Proof of
essentiality is established if no DCOs are obtained in the wildtype background but can be obtained when a functional copy
of the gene is introduced. Further confirmation of essentiality
is made when switching for an empty vector shows that the
integrated copy is required for cell viability, e.g., it cannot be
removed.
13. Typically we use pUC-Hyg-Int for switching individual complementing vectors out of the chromosome. If testing which
genes are essential in an operon, or different gene homologs,
pHAPY1 can be engineered to contain these.
14. In the case of essential genes, there should be no transformants
on the hygromycin plates, and a lawn of growth on the gentamicin. Sometimes the vectors cointegrate into the chromosome, resulting intransformants possessing both Hyg
R and
Gm
R . In this case, typically the numbers of colonies on the
gentamicin plus hygromycin plates and hygromycin only plates
will be similar; if colonies are patched onto gentamicin, they
will be resistant indicating that switching has not occurred. For
nonessential genes, or where the gene being switched in is a
functional homolog, transformants should be obtained on the
298
Amanda Claire Brown