vector can be excised using L5 excisionase (Xis), held on a replicating vector, and essentiality is determined by the inability to obtain
viable transformants [13]. The switching method is described pictorially in Fig. 1. This method has been used successfully for many
essential mycobacterial genes [14, 15].
A further method involves not just the removal of the integrating vector but the switching of it for an empty version of the vector
carrying a different antibiotic selection marker [16]. A diagram of
this methodology is given in Fig. 2. In this case, it has been shown
that excision of the resident vector and integration of the new
plasmid occur at a highly efficient rate. The benefit of this method
is that once a delinquent (Δ’int) strain has been created, any number of different combinations of genes can be switched in and out,
allowing the rapid testing of different versions of the essential gene
Fig. 1 Step-by-step schematic of the gene switching method for identifying essential genes by gene switching: (1) Streak SCO strain (see Note 1) onto a 7H10-KHX plate, after incubation select a single blue
transformant, culture in 10 mL 7H9-Tw-KH for $6d, use this starter culture to inoculate a roller bottle
(100 mL 7H9-Tw-KH for 6d), and prepare cells for electroporation (as described in Subheading 3.1, steps
1–9). (2) Electroporate complementing vector into SCO (as described in Subheading 3.1, steps 10–11).
(3) Select transformants on 7H10-KHGmX. After 2–5 week of incubation at 37
C, streak a single blue
transformant onto 7H10-GmX, and incubate for 1 week at 37
C. (4) Vortex a loopful of the growth from the
7H10-GmX in a 50 mL tube containing 1 mL volume of 1 mm sterile glass beads and 3 mL 7H9-Tw and plate
serial dilutions onto 7H10-GmXSuc; incubate at 37
C for 4–6 weeks. (5) Select 40 white suc
R
transformants
and patch in duplicate onto 7H10-KH and 7H10-GmX. (6) Prepare DNA from white KH sensitive transformants,
PCR screen to identify Δ
0
ints. Confirm with Southern blotting (see Note 11). (7) Electroporate switching vector
into Δ
0
int strain (as described in Subheading 3.3). (8) No viable transformants (Hyg
R
) will be obtained when
trying to switch out an essential gene
Gene Switching and Essentiality Testing
287
viable transformants [13]. The switching method is described pictorially in Fig. 1. This method has been used successfully for many
essential mycobacterial genes [14, 15].
A further method involves not just the removal of the integrating vector but the switching of it for an empty version of the vector
carrying a different antibiotic selection marker [16]. A diagram of
this methodology is given in Fig. 2. In this case, it has been shown
that excision of the resident vector and integration of the new
plasmid occur at a highly efficient rate. The benefit of this method
is that once a delinquent (Δ’int) strain has been created, any number of different combinations of genes can be switched in and out,
allowing the rapid testing of different versions of the essential gene
Fig. 1 Step-by-step schematic of the gene switching method for identifying essential genes by gene switching: (1) Streak SCO strain (see Note 1) onto a 7H10-KHX plate, after incubation select a single blue
transformant, culture in 10 mL 7H9-Tw-KH for $6d, use this starter culture to inoculate a roller bottle
(100 mL 7H9-Tw-KH for 6d), and prepare cells for electroporation (as described in Subheading 3.1, steps
1–9). (2) Electroporate complementing vector into SCO (as described in Subheading 3.1, steps 10–11).
(3) Select transformants on 7H10-KHGmX. After 2–5 week of incubation at 37
C, streak a single blue
transformant onto 7H10-GmX, and incubate for 1 week at 37
C. (4) Vortex a loopful of the growth from the
7H10-GmX in a 50 mL tube containing 1 mL volume of 1 mm sterile glass beads and 3 mL 7H9-Tw and plate
serial dilutions onto 7H10-GmXSuc; incubate at 37
C for 4–6 weeks. (5) Select 40 white suc
R
transformants
and patch in duplicate onto 7H10-KH and 7H10-GmX. (6) Prepare DNA from white KH sensitive transformants,
PCR screen to identify Δ
0
ints. Confirm with Southern blotting (see Note 11). (7) Electroporate switching vector
into Δ
0
int strain (as described in Subheading 3.3). (8) No viable transformants (Hyg
R
) will be obtained when
trying to switch out an essential gene
Gene Switching and Essentiality Testing
287