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 JoVE Biology

माइक्रोबैक्टीरिया की electroporation

1, 1,2

1Center for Infectious Disease, Barts and the London School of Medicine and Dentistry, 2Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry

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    Summary

    Mycobacterial रोगजनक रणनीति बुरा समझा रहते हैं. अधिकांश प्रजातियों की धीमी विकास दर है, सेल की दीवार के अभेद्य प्रकृति, और रोगज़नक़ों के साथ काम करने के खतरों माइक्रोबैक्टीरिया अध्ययन करने के लिए मुश्किल बनाने के लिए और हमारे इन जीवों के गरीब को समझने के लिए काफी हद तक जिम्मेदार हैं. इस वीडियो में हम electroporation की तकनीक है, जो एक संक्षिप्त उच्च विद्युत आवेग को subjecting कोशिकाओं को शामिल करने के लिए डीएनए के प्रवेश की अनुमति प्रदर्शन करेंगे. यह माइक्रोबैक्टीरियल कोशिकाओं में डीएनए को शुरू करने के लिए सबसे व्यापक रूप से इस्तेमाल किया विधि है.

    Date Published: 5/23/2008, Issue 15; doi: 10.3791/761

    Cite this Article

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

    Abstract

    उच्च दक्षता परिवर्तन माइक्रोबैक्टीरिया के अध्ययन में एक प्रमुख सीमा है. जीनस माइकोबैक्टीरियम को बदलने के लिए मुश्किल हो सकता है, यह मुख्य रूप से मोटी और मोमी कोशिका दीवार से कारण होता है, लेकिन तथ्य यह है कि सबसे आणविक तकनीक Escherichia कोलाई और दण्डाणु subtilis के रूप में दूर से संबंधित प्रजातियों के लिए विकसित किया गया है द्वारा जटिल है. इन बाधाओं के बावजूद, माइक्रोबैक्टीरियल plasmids की पहचान की गई है और कई माइक्रोबैक्टीरियल प्रजातियों की डीएनए परिवर्तन अब वर्णित किया गया है है. माइक्रोबैक्टीरिया में डीएनए को शुरू करने के लिए सबसे सफल तरीका electroporation है. कई सफल परिवर्तन करने के लिए पैरामीटर योगदान, इन प्रजातियों / तनाव, बदलने डीएनए की प्रकृति, चयन इस्तेमाल किया मार्कर, मध्यम विकास, और electroporation नाड़ी के लिए शर्तों में शामिल हैं. दोनों धीमी गति से और तेजी से उत्पादक के परिवर्तन के लिए अनुकूलित विधियों यहाँ विस्तृत रहे हैं. अलग माइक्रोबैक्टीरियल प्रजातियों के लिए और विभिन्न चयन मार्कर के साथ परिवर्तन क्षमता रिपोर्ट कर रहे हैं.

    Disclosures

    Comments

    23 Comments

    Can electoporated cells be preserved in glycerol?
    Reply

    Posted by: AnonymousMay 20, 2008, 3:24 PM

    If you mean directly after the pulse, we have never tried this, but I would predict the transformation would be low.
    Reply

    Posted by: AnonymousMay 20, 2008, 3:47 PM

    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

    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: AnonymousAugust 25, 2008, 4:06 PM

    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: AnonymousOctober 28, 2008, 3:10 PM

    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

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

    Posted by: AnonymousFebruary 8, 2010, 4:09 AM

    Its a very good site.Thanks for serving this helpful matters.
    Reply

    Posted by: AnonymousJanuary 5, 2009, 7:06 PM

    I want to know why we do electroporation at elevated temperature for Mycobacterial tuberculosis  
    Reply

    Posted by: AnonymousApril 28, 2009, 10:34 AM

    It has been shown to improve the transformation efficiency.
    Reply

    Posted by: AnonymousApril 28, 2009, 11:44 AM

     I want to know why doing electroporation at elevated temperature for Mycobacterial tuberculosis improves transformation efficiency 
    Reply

    Posted by: AnonymousApril 29, 2009, 2:43 AM

    We do not know.
    Reply

    Posted by: AnonymousFebruary 8, 2010, 4:10 AM

    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: AnonymousSeptember 20, 2010, 10:38 PM

    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: AnonymousSeptember 21, 2010, 2:21 AM

    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: AnonymousNovember 7, 2010, 3:39 PM

    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

    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: AnonymousJune 15, 2011, 6:42 AM

    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: AnonymousSeptember 6, 2011, 2:27 PM

    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: AnonymousOctober 21, 2011, 11:30 PM

    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

    The most likely reason is that the level of expression of your fluorescent protein is not high enough to detect.
    Reply

    Posted by: AnonymousOctober 22, 2011, 8:48 AM

    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

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