6. The rules for selection of the lagging strand sequence, which
depend on the position of the target gene in the chromosome
and its direction of transcription, are shown in Table 2.
7. If the desired base change must include one that creates a T-G,
T-T, or G-G mismatch after annealing of the oligo to the
template strand, modify additional bases within and near the
target codon, but which still encodes the desired amino acid
change in the target protein. Doing so creates a “bubble” of
unmatched bases that is not recognized by the MMR system.
This often can be accomplished by changes in the wobble
positions of both the target codon and its upstream codon
(for an example, see Fig. 2).
8. When working with M. tuberculosis, it is critical to carry out
electroporations within a Class I biosafety cabinet in a BSL3
laboratory in accordance with local safety regulations. If the
samples contain salt or lysed cells, arcing can occur, which can
cause the cover of the cuvette to fly off. One should use an
electroporation device that contains a pulse controller and a
cover for the cuvette holder, make sure the sample is free of salt
(see Note 1), and have disinfectant nearby in case arcing occurs.
9. The reason for this outgrowth period is as follows: if there is
more than one copy of the chromosome per cell at the time of
electroporation, and only one of them is modified, the culture
is grown out to segregate the modified chromosome. However, if one suspects the SNP may confer a growth disadvantage, allow the cells to grow out a shorter period of time
(~16 h).
10. Because of variations in electrocompetence and cell density, it is
advisable to plate out a series of dilutions to be certain to
obtain one plate with at least 24 well-isolated colonies. Typically, we make 1:4, 1:16, and 1:64 dilutions of the outgrowth,
and plate out 300 μL of the undiluted outgrowth and each
dilution.
11. Plates are wrapped in aluminum foil to keep them from drying
out during the 3 weeks of incubation at 37
C.
12. Follow the legal and local regulations for removal of
M. tuberculosis samples from the BSL3 laboratory. We totally
immerse the vials containing M. tuberculosis cells in a metal
bead bath set at 85
C for 50 min. It is important to not heat
the samples at much higher temperatures (or longer periods)
because the cells start to clump (or lyse), and thus will not
perform well as templates for whole cell PCR analysis.
13. The Mismatch Amplification Mutation Assay (or MAMAPCR) was described for use in M. smegmatis by van Kessel
and Hatfull [17]. Primers are designed so that the 3
0 terminal
base of one of the primers matches the single base pair
Oligo-Mediated Recombineering in Mycobacteria
317
depend on the position of the target gene in the chromosome
and its direction of transcription, are shown in Table 2.
7. If the desired base change must include one that creates a T-G,
T-T, or G-G mismatch after annealing of the oligo to the
template strand, modify additional bases within and near the
target codon, but which still encodes the desired amino acid
change in the target protein. Doing so creates a “bubble” of
unmatched bases that is not recognized by the MMR system.
This often can be accomplished by changes in the wobble
positions of both the target codon and its upstream codon
(for an example, see Fig. 2).
8. When working with M. tuberculosis, it is critical to carry out
electroporations within a Class I biosafety cabinet in a BSL3
laboratory in accordance with local safety regulations. If the
samples contain salt or lysed cells, arcing can occur, which can
cause the cover of the cuvette to fly off. One should use an
electroporation device that contains a pulse controller and a
cover for the cuvette holder, make sure the sample is free of salt
(see Note 1), and have disinfectant nearby in case arcing occurs.
9. The reason for this outgrowth period is as follows: if there is
more than one copy of the chromosome per cell at the time of
electroporation, and only one of them is modified, the culture
is grown out to segregate the modified chromosome. However, if one suspects the SNP may confer a growth disadvantage, allow the cells to grow out a shorter period of time
(~16 h).
10. Because of variations in electrocompetence and cell density, it is
advisable to plate out a series of dilutions to be certain to
obtain one plate with at least 24 well-isolated colonies. Typically, we make 1:4, 1:16, and 1:64 dilutions of the outgrowth,
and plate out 300 μL of the undiluted outgrowth and each
dilution.
11. Plates are wrapped in aluminum foil to keep them from drying
out during the 3 weeks of incubation at 37
C.
12. Follow the legal and local regulations for removal of
M. tuberculosis samples from the BSL3 laboratory. We totally
immerse the vials containing M. tuberculosis cells in a metal
bead bath set at 85
C for 50 min. It is important to not heat
the samples at much higher temperatures (or longer periods)
because the cells start to clump (or lyse), and thus will not
perform well as templates for whole cell PCR analysis.
13. The Mismatch Amplification Mutation Assay (or MAMAPCR) was described for use in M. smegmatis by van Kessel
and Hatfull [17]. Primers are designed so that the 3
0 terminal
base of one of the primers matches the single base pair
Oligo-Mediated Recombineering in Mycobacteria
317