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How to prepare Gram(+) bacteria for electrotransformation

Gram(+) bacteria electrocompetent electroporation

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10 replies to this topic

#1 joy123

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Posted 13 February 2012 - 01:48 PM

Hi, I want to transform my plasmid into a G(+) bacteria. I cannot find a standard protocol for preparing the cells. Would you please give me some recommendations?

Thanks a lot!

#2 pDNA

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Posted 14 February 2012 - 10:01 AM

unfortunately, there is no standard protocol available! ...every bug is different and you'll have to optimize your protocol for each species.

What organism are you working with?
For gram+ often protease treatment (S-layer) or treatment with mutanolysin is a good choice!

Regards,
p

#3 joy123

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Posted 28 February 2012 - 01:53 PM

I am using an electroporator for transformation. My bacteria is rarely studied and there is no report how to do transformation on it.

I have tried 1kV,2kV,3kV in 0.1cm cuvette, 4ms. It turned out that the 3kv kills the bateria. 1and 2kV keeps the bacteria alive, but the transformation failed.

Do you have suggestions what I should do next? When should I use protease treatment? And how can I know if my plasmids (I have tried 3 plasmids) are resticted to the bacteria?

Thanks! And look forward to hear from you!


unfortunately, there is no standard protocol available! ...every bug is different and you'll have to optimize your protocol for each species.

What organism are you working with?
For gram+ often protease treatment (S-layer) or treatment with mutanolysin is a good choice!

Regards,
p



#4 pDNA

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Posted 29 February 2012 - 10:58 AM

first of all ...it's not only up to the settings you use for electroporation ...if the cells are not competent they will not uptake any DNA ...so you have to treat em correctly before electroporation as well ...and how you do is up to you and your knowledge of the characteristics of your bug you are working with. If there is no knowledge than you will end up with a trial and error approach (that's the reality for most of the people!).

I would do a literature search for related bacteria ...hopefully you come up with a protocol that i would try. If it fails i would change parameters (one at a time!). Additionally, i would try different pre-treatments like mutanolysin, lysozyme, protease K (one at a time!).

regarding S-layer: you can check if you have an S-layer by running whole cell lysate on an SDS PAGE gel ...the S-layer protein will give you a bright band somewhere around 50 kDa (varies from strain to strain) ...the S-layer can be also extracted (LiCl treatment, various protocols available).

Why do you believe why most of the people prefere E. coli? ...because it simple works :)
Starting to work with a new bug (especially gram positive) is really hard ...since you don't know nothing ...establishing a transformation protocol is plain hard work ...and nowadays nobody wants to do those things ...because you hardly get those things published (most reviewers think this is too "simple").

So don't worry and try hard :)

Good luck!

Regards,
p

#5 mdfenko

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Posted 29 February 2012 - 12:32 PM

i don't know the specific protocol because i have never personally done it but can you try making them competent chemically (with calcium chloride)?

the protocol should be in sambrook.
talent does what it can
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#6 joy123

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Posted 29 February 2012 - 02:02 PM

Hi! Thanks very much for your kind help! I will start with reading about similar bacteria. Thanks again!

first of all ...it's not only up to the settings you use for electroporation ...if the cells are not competent they will not uptake any DNA ...so you have to treat em correctly before electroporation as well ...and how you do is up to you and your knowledge of the characteristics of your bug you are working with. If there is no knowledge than you will end up with a trial and error approach (that's the reality for most of the people!).

I would do a literature search for related bacteria ...hopefully you come up with a protocol that i would try. If it fails i would change parameters (one at a time!). Additionally, i would try different pre-treatments like mutanolysin, lysozyme, protease K (one at a time!).

regarding S-layer: you can check if you have an S-layer by running whole cell lysate on an SDS PAGE gel ...the S-layer protein will give you a bright band somewhere around 50 kDa (varies from strain to strain) ...the S-layer can be also extracted (LiCl treatment, various protocols available).

Why do you believe why most of the people prefere E. coli? ...because it simple works Posted Image
Starting to work with a new bug (especially gram positive) is really hard ...since you don't know nothing ...establishing a transformation protocol is plain hard work ...and nowadays nobody wants to do those things ...because you hardly get those things published (most reviewers think this is too "simple").

So don't worry and try hard Posted Image

Good luck!

Regards,
p



#7 joy123

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Posted 29 February 2012 - 02:05 PM

Thanks for your response. I thought the electrotransfortation may work better than chemical transformation, and G(+) is more tough than G(-), so I started with electro-transformation. I prepared competent cells according to online protocols, but I am not sure if it is suitable for my bacteria. Please let me know if you have further question. Thanks a lot!

,

i don't know the specific protocol because i have never personally done it but can you try making them competent chemically (with calcium chloride)?

the protocol should be in sambrook.



#8 pDNA

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Posted 29 February 2012 - 02:09 PM

you could try for example Lithiumacetate:
http://www.biomedcen.../1472-6750/7/15

chemical competent cells won't help much ...you'll have to stick to electrotransformation!

Regards,
p

#9 joy123

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Posted 04 March 2012 - 07:57 AM

Thanks a lot! Now I have a question: I don't know what concentration of antibiotics should I use for selecting the transformed bacteria after electroporation. Should I do an experiment to find the MIC (minimun inhibitory concentration), and use the MIC (or higher than MIC?) in the agar culture media? Besides, should I use normal bacteria or electroporated bacteria for testing MIC?

Thanks very much!



you could try for example Lithiumacetate:
http://www.biomedcen.../1472-6750/7/15

chemical competent cells won't help much ...you'll have to stick to electrotransformation!

Regards,
p



#10 phage434

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Posted 04 March 2012 - 07:32 PM

I would use untransformed cells to test the MIC, then use about 3x MIC in plates for selection. MIC is easy to measure. Infect media with your culture, then aliquot 100 ul into a row of wells on a plate. Add antibiotic to media at a high known concentration, then add 100 ul to the leftmost well. Do 2x serial dilutions across the row, leaving the last well untouched (zero antibiotic). Cover, incubate, and observe growth.

#11 joy123

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Posted 06 March 2012 - 01:04 PM

Thanks a lot! I have prepared 3XMIC today, and will see how the transformation works.

I would use untransformed cells to test the MIC, then use about 3x MIC in plates for selection. MIC is easy to measure. Infect media with your culture, then aliquot 100 ul into a row of wells on a plate. Add antibiotic to media at a high known concentration, then add 100 ul to the leftmost well. Do 2x serial dilutions across the row, leaving the last well untouched (zero antibiotic). Cover, incubate, and observe growth.







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