reduced transformation efficiency. During the standing time on
ice prior to pulse delivery, the cells may settle in the tube and it
is necessary to redistribute them using a pipette or a vortex
immediately prior to the high-voltage pulse. This step serves to
resuspend the cells and to insure thorough mixing of the DNA.
In addition, care must be taken to insure that no bubbles are
introduced between the two electrodes of the cuvette.
References
1. Jacobs WR, Kalpana GV, Cirillo JD et al (1991)
Genetic Systems for Mycobacteria. Methods
Enzymol 204:537–555. https://doi.org/10.
1016/0076-6879(91)04027-L
2. Kalpana GV, Bloom BR, Jacobs WR (1991)
Insertional mutagenesis and illegitimate
recombination in mycobacteria. Proc Natl
Acad Sci U S A 88:5433–5437. https://doi.
org/10.1073/pnas.88.12.5433
3. Dellagostin OA, Wall S, Norman E et al (1993)
Construction and use of integrative vectors to
express foreign genes in mycobacteria. Mol
Microbiol 10:983–993. https://doi.org/10.
1111/j.1365-2958.1993.tb00970.x
4. Garbe TR, Barathi J, Barnini S et al (1994)
Transformation of mycobacterial species using
hygromycin resistance as selectable marker.
Microbiology 140:133–138. https://doi.org/
10.1099/13500872-140-1-133
5. Qin M, Taniguchi H, Mizuguchi Y (1994)
Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol
176:419–425. https://doi.org/10.1128/jb.
176.2.419-425.1994
6. Hermans J, Martin C, Huijberts GN et al
(1991) Transformation of Mycobacterium
aurum and Mycobacterium smegmatis with
the broad host-range gram-negative cosmid
vector
pJRD215.
Mol
Microbiol
5:1561–1566.
https://doi.org/10.1111/j.
1365-2958.1991.tb00803.x
7. Wards BJ, Collins DM (1996) Electroporation
at elevated temperatures substantially improves
transformation efficiency of slow-growing
mycobacteria.
FEMS
Microbiol
Lett
145:101–105.
https://doi.org/10.1111/j.
1574-6968.1996.tb08563.x
8. Talaat AM, Trucksis M (2000) Transformation
and transposition of the genome of Mycobacterium marinum. Am J Vet Res 61:125–128.
https://doi.org/10.2460/ajvr.2000.61.125
9. Beggs ML, Crawford JT, Eisenach KD (1995)
Isolation and sequencing of the replication
region of Mycobacterium avium plasmid
pLR7. J Bacteriol 177:4836–4840. https://
doi.org/10.1128/jb.177.17.4836-4840.
1995
10. Lee S-H, Cheung M, Irani V et al (2002) Optimization of electroporation conditions for
Mycobacterium avium. Tuberculosis (Edinb)
82:167–174. https://doi.org/10.1054/tube.
2002.0335
11. Paget E, Davies J (1996) Apramycin resistance
as a selective marker for gene transfer in mycobacteria. J Bacteriol 178:6357–6360. https://
doi.org/10.1128/jb.178.21.6357-6360.
1996
12. Ranes MG, Rauzier J, Lagranderie M et al
(1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia
coli
shuttle
vector.
J
Bacteriol
172:2793–2797. https://doi.org/10.1128/
jb.172.5.2793-2797.1990
13. Bachrach G, Colston MJ, Bercovier H et al
(2000) A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum
which is compatible with the pAL5000 replicon The EMBL accession number for the
sequence determined in this work is
AJ238973.
Microbiology
146:297–303.
https://doi.org/10.1099/00221287-146-2297
14. Gavigan JA, Aı ´nsa JA, Pe ´rez E et al (1997)
Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium
fortuitum. J Bacteriol 179:4115–4122.
https://doi.org/10.1128/jb.179.13.41154122.1997
15. Gormley EP, Davies J (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia
coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173:6705–6708.
https://doi.org/10.1128/jb.173.21.67056708.1991
16. Stover CK, de la Cruz VF, Fuerst TR et al
(1991) New use of BCG for recombinant vaccines. Nature 351:456–460. https://doi.org/
10.1038/351456a0
Electroporation
283
ice prior to pulse delivery, the cells may settle in the tube and it
is necessary to redistribute them using a pipette or a vortex
immediately prior to the high-voltage pulse. This step serves to
resuspend the cells and to insure thorough mixing of the DNA.
In addition, care must be taken to insure that no bubbles are
introduced between the two electrodes of the cuvette.
References
1. Jacobs WR, Kalpana GV, Cirillo JD et al (1991)
Genetic Systems for Mycobacteria. Methods
Enzymol 204:537–555. https://doi.org/10.
1016/0076-6879(91)04027-L
2. Kalpana GV, Bloom BR, Jacobs WR (1991)
Insertional mutagenesis and illegitimate
recombination in mycobacteria. Proc Natl
Acad Sci U S A 88:5433–5437. https://doi.
org/10.1073/pnas.88.12.5433
3. Dellagostin OA, Wall S, Norman E et al (1993)
Construction and use of integrative vectors to
express foreign genes in mycobacteria. Mol
Microbiol 10:983–993. https://doi.org/10.
1111/j.1365-2958.1993.tb00970.x
4. Garbe TR, Barathi J, Barnini S et al (1994)
Transformation of mycobacterial species using
hygromycin resistance as selectable marker.
Microbiology 140:133–138. https://doi.org/
10.1099/13500872-140-1-133
5. Qin M, Taniguchi H, Mizuguchi Y (1994)
Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol
176:419–425. https://doi.org/10.1128/jb.
176.2.419-425.1994
6. Hermans J, Martin C, Huijberts GN et al
(1991) Transformation of Mycobacterium
aurum and Mycobacterium smegmatis with
the broad host-range gram-negative cosmid
vector
pJRD215.
Mol
Microbiol
5:1561–1566.
https://doi.org/10.1111/j.
1365-2958.1991.tb00803.x
7. Wards BJ, Collins DM (1996) Electroporation
at elevated temperatures substantially improves
transformation efficiency of slow-growing
mycobacteria.
FEMS
Microbiol
Lett
145:101–105.
https://doi.org/10.1111/j.
1574-6968.1996.tb08563.x
8. Talaat AM, Trucksis M (2000) Transformation
and transposition of the genome of Mycobacterium marinum. Am J Vet Res 61:125–128.
https://doi.org/10.2460/ajvr.2000.61.125
9. Beggs ML, Crawford JT, Eisenach KD (1995)
Isolation and sequencing of the replication
region of Mycobacterium avium plasmid
pLR7. J Bacteriol 177:4836–4840. https://
doi.org/10.1128/jb.177.17.4836-4840.
1995
10. Lee S-H, Cheung M, Irani V et al (2002) Optimization of electroporation conditions for
Mycobacterium avium. Tuberculosis (Edinb)
82:167–174. https://doi.org/10.1054/tube.
2002.0335
11. Paget E, Davies J (1996) Apramycin resistance
as a selective marker for gene transfer in mycobacteria. J Bacteriol 178:6357–6360. https://
doi.org/10.1128/jb.178.21.6357-6360.
1996
12. Ranes MG, Rauzier J, Lagranderie M et al
(1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia
coli
shuttle
vector.
J
Bacteriol
172:2793–2797. https://doi.org/10.1128/
jb.172.5.2793-2797.1990
13. Bachrach G, Colston MJ, Bercovier H et al
(2000) A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum
which is compatible with the pAL5000 replicon The EMBL accession number for the
sequence determined in this work is
AJ238973.
Microbiology
146:297–303.
https://doi.org/10.1099/00221287-146-2297
14. Gavigan JA, Aı ´nsa JA, Pe ´rez E et al (1997)
Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium
fortuitum. J Bacteriol 179:4115–4122.
https://doi.org/10.1128/jb.179.13.41154122.1997
15. Gormley EP, Davies J (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia
coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173:6705–6708.
https://doi.org/10.1128/jb.173.21.67056708.1991
16. Stover CK, de la Cruz VF, Fuerst TR et al
(1991) New use of BCG for recombinant vaccines. Nature 351:456–460. https://doi.org/
10.1038/351456a0
Electroporation
283