Skip to main content
SpringerLink
Log in

Electroporation of Mycobacteria

  • Protocol
  • First Online:
Mycobacteria Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1285))

Abstract

High-efficiency transformation of DNA is integral to the study of mycobacteria, allowing genetic manipulation. Electroporation is the most widely used method for introducing DNA into mycobacterial strains. Many parameters contribute to high-efficiency transformation; these include the species per strain, the transforming DNA, the selectable marker, the growth medium additives, and the conditions of electroporation. In this chapter we provide an optimized method for the transformation of representative slow- and fast-growing species of mycobacteria—Mycobacterium tuberculosis and M. smegmatis, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Jacobs WR Jr, Kalpana GV, Cirillo JD, Pascopella L, Snapper SB, Udani RA, Jones W, Barletta RG, Bloom BR (1991) Genetic systems for mycobacteria. Methods Enzymol 204:537–555

    Article  CAS  PubMed  Google Scholar 

  2. Kalpana GV, Bloom BR, Jacobs WR Jr (1991) Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc Natl Acad Sci U S A 88(12):5433–5437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Paget E, Davies J (1996) Apramycin resistance as a selective marker for gene transfer in mycobacteria. J Bacteriol 178(21):6357–6360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Consaul SA, Pavelka MS Jr (2004) Use of a novel allele of the Escherichia coli aacC4 aminoglycoside resistance gene as a genetic marker in mycobacteria. FEMS Microbiol Lett 234(2):297–301

    Article  CAS  PubMed  Google Scholar 

  5. Parish T, Gordhan BG, McAdam RA, Duncan K, Mizrahi V, Stoker NG (1999) Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology 145(Pt 12):3497–3503

    Article  CAS  PubMed  Google Scholar 

  6. Ranes MG, Rauzier J, Lagranderie M, Gheorghiu M, Gicquel B (1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia coli shuttle vector. J Bacteriol 172(5):2793–2797

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yuan Y, Crane DD, Simpson RM, Zhu YQ, Hickey MJ, Sherman DR, Barry CE 3rd (1998) The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci U S A 95(16):9578–9583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dellagostin OA, Wall S, Norman E, O’Shaughnessy T, Dale JW, McFadden J (1993) Construction and use of integrative vectors to express foreign genes in mycobacteria. Mol Microbiol 10(5):983–993

    Article  CAS  PubMed  Google Scholar 

  9. Goto Y, Taniguchi H, Udou T, Mizuguchi Y, Tokunaga T (1991) Development of a new host vector system in mycobacteria. FEMS Microbiol Lett 67(3):277–282

    Article  CAS  PubMed  Google Scholar 

  10. Matsuo K, Yamaguchi R, Yamazaki A, Tasaka H, Terasaka K, Totsuka M, Kobayashi K, Yukitake H, Yamada T (1990) Establishment of a foreign antigen secretion system in mycobacteria. Infect Immun 58(12):4049–4054

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Qin M, Taniguchi H, Mizuguchi Y (1994) Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol 176(2):419–425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Radford AJ, Hodgson AL (1991) Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid 25(2):149–153

    Article  CAS  PubMed  Google Scholar 

  13. Snapper SB, Lugosi L, Jekkel A, Melton RE, Kieser T, Bloom BR, Jacobs WR Jr (1988) Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A 85(18):6987–6991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Garbe TR, Barathi J, Barnini S, Zhang Y, Abou-Zeid C, Tang D, Mukherjee R, Young DB (1994) Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology 140:133–138

    Article  CAS  PubMed  Google Scholar 

  15. Wards BJ, Collins DM (1996) Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol Lett 145(1):101–105

    Article  CAS  PubMed  Google Scholar 

  16. Hermans J, Martin C, Huijberts GN, Goosen T, de Bont JA (1991) Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215. Mol Microbiol 5(6):1561–1566

    Article  CAS  PubMed  Google Scholar 

  17. Houssaini-Iraqui M, Lazraq MH, Clavel-Seres S, Rastogi N, David HL (1992) Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis. FEMS Microbiol Lett 69(3):239–244

    Article  CAS  PubMed  Google Scholar 

  18. Marklund BI, Speert DP, Stokes RW (1995) Gene replacement through homologous recombination in Mycobacterium intracellulare. J Bacteriol 177(21):6100–6105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hermans J, Suy IML, De Bont JAM (1993) Transformation of Gram-positive microorganisms with the Gram-negative broad-host-range cosmid vector pJRD215. FEMS Microbiol Lett 108:201–204

    Article  CAS  Google Scholar 

  20. Talaat AM, Trucksis M (2000) Transformation and transposition of the genome of Mycobacterium marinum. Am J Vet Res 61(2):125–128

    Article  CAS  PubMed  Google Scholar 

  21. Beggs ML, Crawford JT, Eisenach KD (1995) Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7. J Bacteriol 177(17):4836–4840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Foley-Thomas EM, Whipple DL, Bermudez LE, Barletta RG (1995) Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology 141:1173–1181

    Article  CAS  PubMed  Google Scholar 

  23. Lee SH, Cheung M, Irani V, Carroll JD, Inamine JM, Howe WR, Maslow JN (2002) Optimization of electroporation conditions for Mycobacterium avium. Tuberculosis (Edinb) 82(4–5):167–174

    Article  Google Scholar 

  24. Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4(11):1911–1919

    Article  CAS  PubMed  Google Scholar 

  25. Labidi A, Dauguet C, Goh KS, David HL (1984) Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol 11:235–240

    Article  CAS  Google Scholar 

  26. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF et al (1991) New use of BCG for recombinant vaccines. Nature 351(6326):456–460

    Article  CAS  PubMed  Google Scholar 

  27. Bachrach G, Colston MJ, Bercovier H, Bar-Nir D, Anderson C, Papavinasasundaram KG (2000) A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146(Pt 2):297–303

    Article  CAS  PubMed  Google Scholar 

  28. Gavigan JA, Ainsa JA, Perez E, Otal I, Martin C (1997) Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum. J Bacteriol 179(13):4115–4122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lee MH, Pascopella L, Jacobs WR Jr, Hatfull GF (1991) Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc Natl Acad Sci U S A 88(8):3111–3115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Anes E, Portugal I, Moniz-Pereira J (1992) Insertion into the Mycobacterium smegmatis genome of the aph gene through lysogenization with the temperate mycobacteriophage Ms6. FEMS Microbiol Lett 74:21–25

    Article  CAS  PubMed  Google Scholar 

  31. England PM, Mazodier P, Mediola MV, Gicquel B, Smokvina T, Thompson CJ, Davies J (1991) Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol Microbiol 5:2499–2502

    Article  Google Scholar 

  32. Bottger EC (1994) Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol 2(10):416–421

    Article  CAS  PubMed  Google Scholar 

  33. Hatfull GF (1993) Genetic transformation of mycobacteria. Trends Microbiol 1(8):310–314

    Article  CAS  PubMed  Google Scholar 

  34. Kana BD, Mizrahi V (2004) Molecular genetics of Mycobacterium tuberculosis in relation to the discovery of novel drugs and vaccines. Tuberculosis (Edinb) 84(1–2):63–75

    Article  Google Scholar 

  35. Aldovini A, Husson RN, Young RA (1993) The uraA locus and homologous recombination in Mycobacterium bovis BCG. J Bacteriol 175(22):7282–7289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Husson RN, James BE, Young RA (1990) Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 172(2):519–524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hermans J, Boschloo JG, de Bont JAM (1990) Transformation of Mycobacterium aurum by electroporation: the use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency. FEMS Microbiol Lett 72:221–224

    Article  CAS  Google Scholar 

  38. Hammes W, Schleifer KH, Kandler O (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol 116(2):1029–1053

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Cruickshank R (1965) Medical microbiology: a guide to the laboratory diagnosis and control of infection, 11th edn. E & S Livingstone Limited, London

    Google Scholar 

  40. David M, Lubinsky-Mink S, Ben-Zvi A, Ulitzur S, Kuhn J, Suissa M (1992) A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin. Plasmid 28(3):267–271

    Article  CAS  PubMed  Google Scholar 

  41. Gormley EP, Davies J (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173(21):6705–6708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett 229(2):211–215

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. University of Rennes, Campus scientifique de Beaulieu, Rennes, France

    Renan Goude

  2. TB Discovery Research Group, Infectious Disease Research Institute, Seattle, WA, 98102, USA

    David M. Roberts & Tanya Parish

Authors
  1. Renan Goude
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David M. Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tanya Parish
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tanya Parish .

Editor information

Editors and Affiliations

  1. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    Tanya Parish

  2. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    David M. Roberts

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Goude, R., Roberts, D.M., Parish, T. (2015). Electroporation of Mycobacteria. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2450-9_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2449-3

  • Online ISBN: 978-1-4939-2450-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation