1. 7H9-AD-tween medium can be used in place of 7H9-OADCtween.
2. Lysogeny broth (LB) plates can be used in place of 7H10OADC plates.
3. ATc induction period: Induce at an OD 600 of 0.5, and grow for
3 h at 37
C with shaking, or until the culture reaches an
OD 600 of 1.0.
4. Keep cells on ice while making them electrocompetent; as with
the M. tuberculosis ORBIT protocol, electroporation cuvettes
do not need to be cooled on ice.
5. Outgrowth period: overnight for both SNP transfer and
ORBIT. However, plating 4 h after electroporation is an option
with both protocols.
6. The addition of 2 M glycine 8 h after ATc induction is not
performed with M. smegmatis.
4 Notes
1. When electroporated into recombinogenic mycobacteria containing pKM427, this oligo will generate hygromycin-resistant
transformants. These Hyg
R transformants are then screened for
incorporation of the SNP-containing oligo.
2. This step helps to remove residual salts from the DNA sample
that can interfere with the efficiency of electroporation. A time
constant of >20 ms should be expected for salt-free samples
using 0.2 cm cuvettes. A time constant of <18 ms is indicative
of salt contamination, which will result in lower efficiencies of
DNA transfer across the membrane.
3. While plasmid pKM402 is described here for use in transferring
SNPs to the mycobacterial chromosome, pKM461 could also
be used, as both plasmids express RecT from an Atc-inducible
promoter. While pKM402 has been principally employed in
this lab for the development of the SNP transfer protocols
described in this chapter, plasmid pKM461 has the advantage
that it can be cured from the recombinant by counter-selection
on 7H10-sucrose plates following SNP transfer, if required.
The expression of Bxb1 Integrase from pKM461 does not
interfere with SNP transfer.
4. For more electroporations, set up additional cultures.
5. Even though frozen cells are adequate for transformation of
replicating/integrating plasmids, freshly prepared electrocompetent cells are required for generating mutants by
recombineering.
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Kenan C. Murphy
2. Lysogeny broth (LB) plates can be used in place of 7H10OADC plates.
3. ATc induction period: Induce at an OD 600 of 0.5, and grow for
3 h at 37
C with shaking, or until the culture reaches an
OD 600 of 1.0.
4. Keep cells on ice while making them electrocompetent; as with
the M. tuberculosis ORBIT protocol, electroporation cuvettes
do not need to be cooled on ice.
5. Outgrowth period: overnight for both SNP transfer and
ORBIT. However, plating 4 h after electroporation is an option
with both protocols.
6. The addition of 2 M glycine 8 h after ATc induction is not
performed with M. smegmatis.
4 Notes
1. When electroporated into recombinogenic mycobacteria containing pKM427, this oligo will generate hygromycin-resistant
transformants. These Hyg
R transformants are then screened for
incorporation of the SNP-containing oligo.
2. This step helps to remove residual salts from the DNA sample
that can interfere with the efficiency of electroporation. A time
constant of >20 ms should be expected for salt-free samples
using 0.2 cm cuvettes. A time constant of <18 ms is indicative
of salt contamination, which will result in lower efficiencies of
DNA transfer across the membrane.
3. While plasmid pKM402 is described here for use in transferring
SNPs to the mycobacterial chromosome, pKM461 could also
be used, as both plasmids express RecT from an Atc-inducible
promoter. While pKM402 has been principally employed in
this lab for the development of the SNP transfer protocols
described in this chapter, plasmid pKM461 has the advantage
that it can be cured from the recombinant by counter-selection
on 7H10-sucrose plates following SNP transfer, if required.
The expression of Bxb1 Integrase from pKM461 does not
interfere with SNP transfer.
4. For more electroporations, set up additional cultures.
5. Even though frozen cells are adequate for transformation of
replicating/integrating plasmids, freshly prepared electrocompetent cells are required for generating mutants by
recombineering.
316
Kenan C. Murphy