Electroporation of Mycobacteria

Biology
 

Summary

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Cite this Article

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Goude, R., Parish, T. Electroporation of Mycobacteria. J. Vis. Exp. (15), e761, doi:10.3791/761 (2008).

Abstract

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Disclosures

The authors have nothing to disclose.

Comments

26 Comments

  1. Can electoporated cells be preserved in glycerol?

    Reply
    Posted by: Anonymous
    May 20, 2008 - 3:24 PM
  2. If you mean directly after the pulse, we have never tried this, but I would predict the transformation would be low.

    Reply
    Posted by: Anonymous
    May 20, 2008 - 3:47 PM
  3. Do you mean the cells just after electoporation? We had never tried that. However, you can preserve the competent cell in glycerol, though the effiiciay would be lower.

    Reply
    Posted by: Nan Z.
    October 18, 2010 - 11:32 AM
  4. Hell I wanted to know if it would be possible to get a printable copy of the paper. I have some questions about the mediums in general. I appreciate your help!

    Reply
    Posted by: Anonymous
    August 25, 2008 - 4:06 PM
  5. Hell I wanted to know if it would be possible to get a printable copy of the paper. I have some questions about the mediums in general. I appreciate your help!

    Reply
    Posted by: Anonymous
    October 28, 2008 - 3:10 PM
  6. I am trying to transform BCG by your protocol electroporation, but unfortunately we cannot find recombinant BCG colonies until now. What is the critical point in this protocol? Would you please send me a printable copy of the paper?

    Reply
    Posted by: maryam y.
    February 6, 2010 - 1:27 AM
  7. The cells must be actively and aerobically growing before transformation. As far as we can ascertain, there is no critical point in the protocol, we always follow the whole protocol to obtain good efficiencies. Have you made any modifications to the method?

    I am afraid there is no printable copy of this article, it is by nature a video.

    Reply
    Posted by: Anonymous
    February 8, 2010 - 4:08 AM
  8. I am afraid there is no printable copy of this article, it is by nature a video.

    Reply
    Posted by: Anonymous
    February 8, 2010 - 4:09 AM
  9. Its a very good site.Thanks for serving this helpful matters.

    Reply
    Posted by: Anonymous
    January 5, 2009 - 7:06 PM
  10. I want to know why we do electroporation at elevated temperature for Mycobacterial tuberculosis  

    Reply
    Posted by: Anonymous
    April 28, 2009 - 10:34 AM
  11. It has been shown to improve the transformation efficiency.

    Reply
    Posted by: Anonymous
    April 28, 2009 - 11:44 AM
  12.  I want to know why doing electroporation at elevated temperature for Mycobacterial tuberculosis improves transformation efficiency 

    Reply
    Posted by: Anonymous
    April 29, 2009 - 2:43 AM
  13. We do not know.

    Reply
    Posted by: Anonymous
    February 8, 2010 - 4:10 AM
  14. Can I prepare electrocompetent from direct colonies (pick a lot of colonies on LJ to vortex with glass beads and prepare electrocompetent from that)?

    Reply
    Posted by: Anonymous
    September 20, 2010 - 10:38 PM
  15. We have never tried this, but I would be concerned about having clumps of cells even after vortexing and that would lead to arcing.

    Reply
    Posted by: Anonymous
    September 21, 2010 - 2:21 AM
  16. I'm studying in master's degree program and try (be tried) to transfer recombinant plasmid to Mtb H37Ra (modify from your protocol). I used kanamycin as selective marker and added amphotericin B + vancomycin to M7H11 but I still found fungal contamination. Question is how you managed your plate after spreaded? May you have some technique that not write in "Mycobacteria protocols".
    (I'm not seal plate, put them in plastic bag and bring them to 37C incubator.)

    Reply
    Posted by: Anonymous
    November 7, 2010 - 3:39 PM
  17. Some fungi are resistive to amphotericin B, seal your plate with preservative film after spreaded and store them to 4C frig.

    Reply
    Posted by: Nan Z.
    November 9, 2010 - 7:00 AM
  18. DŒs pretreatment of BCG with glycine help in increasing the transformation efficiency? Also how long can you grow BCG in presence of glycine before harvesting them?

    Reply
    Posted by: Anonymous
    June 15, 2011 - 6:42 AM
  19. It would be useful to my master Thesis, I am trying to knock out a gene, In this moment I am preparing my electrocompetent cells, next week I will perform the electroporation and I will back with some questions. thanks Dr.Tanya for your mail. greetings since Peru.

    Reply
    Posted by: Anonymous
    September 6, 2011 - 2:27 PM
  20. We are trying to integrate a RFP gene in pMV361 into Mtb H37Rv. After electropoaration we did get colonies on Kan plate, but the bacilli were not fluorescent unlike the ones we got after electropoartion with pMV²61-RFP. What could be the reason?

    Reply
    Posted by: Anonymous
    October 21, 2011 - 11:30 PM
  21. Have you ensured that the plasmid existed in the obtained transformants? I mean theat the colonies maybe false positive. If your transformants did contain the plasmid, there maybe something wrong with the expression of the RFP protein. For example, the promoter of the RFP is not strong enough, thus the fluorescence could not be detected.

    Reply
    Posted by: Nan Z.
    October 24, 2011 - 2:46 AM
  22. The most likely reason is that the level of expression of your fluorescent protein is not high enough to detect.

    Reply
    Posted by: Anonymous
    October 22, 2011 - 8:48 AM
  23. Dear Sir,

    I have tried to electroporate M. smegmatis mc² 155 today by following this video and the protocols from your book, Mycobacteria protocols, ²nd edition. However, the time constant is quite low, just 6.9 ms so I guess my electroporation is likely unsuccessful. I were using the 7H9 medium to culture cells. Have you had any comments and suggestion for the low time constant and how I can improve this?

    Thank you very much!

    Reply
    Posted by: Khanh N.
    June 6, 2012 - 3:08 AM
  24. Hi, I would like to know whether centrifugation and handling steps (for cell harvesting and washing) are to be performed at 4 degrees celsius or RT for electroporation of M. smegmatis.

    Reply
    Posted by: Yoganand K.
    January 9, 2015 - 1:16 AM
  25. Hello, I am trying to do electroporation based on this video for mycobacterium marinum. I am having hard time in getting growth with OD 1 even after 5 days of incubation. Should I allow the bacteria to grow until I get the OD of 1 or should I grow the bacteria only for 24 hrs and do electroporation?

    Reply
    Posted by: deepti p.
    May 11, 2015 - 12:35 PM
  26. surely you can't actually mean 300 x g? Did you mean 3000 x g?

    Reply
    Posted by: jordan h.
    April 7, 2016 - 3:20 PM

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