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Electroporation for Marine bacteria, Help me!


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#1 shomi83

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Posted 23 June 2011 - 06:21 PM

Hi friends,

I am now planing to do transposon mutagenesis. You know that electroporation is an effective to introduce heterologous gene into host cells. My bacteria is marine bacteria so that I now jump in some problems. Before conducting electroporation, salt must be removed completedly. My bacteria cannot survive in none-salt environment. I am trying to modify some conditions. I read some paper and they use 10mM Mg2+ solution to wash the cells instead of deionized water. But the last step, cells must be resuspended in 10% glycerol. I ever tried to solve cells in 10% glycerol and spread on agar plate. But no colony, I guess the cell was lysed in 10% glycerol solution.

I hope you guys can help me to solve this problem. How can I apply electroporation for marine bacteria?

#2 gyma

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Posted 24 June 2011 - 01:16 AM

I dont think glycerol would kill your bacteria because we use glycerol solution to store E.coli at -80c. might be the problem of your Mg2+ solution or other washing procedures.
I did electroporation before and I did have the problem of arcing because of the salt in my plasmid DNA. Competent E.coli is fine and doesnt have a issue of salt. I dont get it very clear by what you said. Your bacteria would die without salt but does it happen very fast? because you wont spend hours in electroporation, I think less than 30 mins you could get the job done.

#3 Adrian K

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Posted 24 June 2011 - 02:09 AM

How do you make your 10% glycerol? Just curious...
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

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#4 phage434

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Posted 24 June 2011 - 06:32 AM

You can make high osmolarity solutions with sucrose or sorbitol which may help in your transformation. 250-500 mM sucrose might be a good place to start. There is probably literature on this, but I'm not familiar with it. Sites such as Epicentre have lists of organisms and papers with successful transposon insertions via electroporation. Biorad and BTK have lists of electroporation conditions for different organisms.

#5 shomi83

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Posted 01 July 2011 - 05:24 AM

Thanks for your comments.

My problem here is that the bacteria I am working with is Pseudoalteromonas sp. isolated from seawater. For preparing growth medium, I use seawater. I think you know the condition for successful electroporation is to remove completedly salt by washing the cells in ddH20 containing 10% glycerol before starting. I read some papers and see that some buffers containing sucrose 252mM, MgSO4, glycerol were applied well for marine bacteria. I tried to screen the viability of my bacteria by taking colony from agar plate and dissolve colonies into these electroporation buffer. After that, I spread this suspension on agar plate and incubate at 20oC (the optimal degree for my bacteria). But after incubation, I didnot see any colonies appeared on agar plate. I guess my bacteia were lysed when I suspended in the poration buffer. So If my bacteria could not survive after treatment for electroporation, I cannot do electroporation then. Actually, until now I already try with ten kinds of electroporation buffer. But I still not see any colony on agar plate after treatment the cells.
Could you give any susgestion?

#6 shomi83

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Posted 01 July 2011 - 05:29 AM

How do you make your 10% glycerol? Just curious...

I dissolve 1V of glycerol into 9V of ddH2O and autoclave. Do you think it is no problem?

#7 Adrian K

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Posted 03 July 2011 - 07:14 AM


How do you make your 10% glycerol? Just curious...

I dissolve 1V of glycerol into 9V of ddH2O and autoclave. Do you think it is no problem?


Correct me if I'm wrong. I got not experience in your field but...
I think you should not use ddH2O in your 10% glycerol stock preparation. This might be the reason for the osmolarity changes which killed your bacteria. You can directly plate your bacteria after 10% glycerol wash and see if your bacteria is still viable. As phage434 pointed out, you can use sucrose as a replacement. Perhaps try to use up to 10% sucrose? (But I really unsure whether if this works for electroporation, I really don't know)

However, according to this article: http://jvi.asm.org/c.../80/18/9270.pdf
The author uses:
7 mM HEPES [pH 7.0], 252 mM sucrose, 20% glycerol
as washing buffer for Pseudoalteromons sp.

If is not working, write to the author... ^_^
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#8 Nithin

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Posted 03 December 2013 - 09:10 PM

Hello,

 

Did you manage to solve your issue with the electroporation of marine bacteria?

 

I am facing the same problem with my marine strains. However my cells dont lyse when grown in LB in ddH20 and resuspended n 10% glycerol. I have plated them several times and checked and they give a nice lawn culture. 

 

However I do no get any transformants when I do electroporation. I have checked under various conditions and it still does not work. Any suggestions??

 

Thanks

Nithin






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