problems with electroporation of Listeria - please, if you have any ideas.. (Jun/09/2011 )

Hi, all!

I am trying to transform Listeria monocytogenes EGD with a plasmid for over a 6 months and really need some advice because I am not getting ANY transformed bacteria at all.

I tried several techniques, now I am doing the electroporation according to Monk et al., 2008 (http://aem.asm.org/cgi/reprint/74/13/3921), because they stated that by means of Park and Stewart method EGD is poorly transformed (I did not get anything), and yet do not observe any colonies. Ctrls (bacteria transformed with water instead of plasmid) are growing nicely, though a bit smaller than usually.
Did anybody try this method, or it has some other method that works for him? What are crucial points in transformation of L.m. EGD that could effect it? I am also working with E.coli and it is really easy to transform it, I got a lot of colonies both heat-shock and electroporation method, so this L.m. EGD issues are driving me crazy..

Pretty please, if anybody has any idea..

Thank you very-very-very much!!!

Have you paid any attention to the restriction systems native in the organism? Elimination of restriction sites, or methylation of them with appropriate methylases may solve your problem. Are you certain your resistance cassettes function in L.m.? Have you tried a control transformation with a plasmid known to function in the species?

phage434 on Thu Jun 9 23:48:34 2011 said:

Actually, I did not do that. I cannot find any references regarding restriction systems native in Listeria monocytogenes. Is there any program I can use to check that or how is it done?

phage434 on Thu Jun 9 23:48:34 2011 said:

I am using erythromicin cassette that was already used before in other construct tranformed in L.m.

phage434 on Thu Jun 9 23:48:34 2011 said:

Yes, I have tried it (got it recently as a gift), but still not working, so I am desperate..




Thank you for your kind ideas and help

So, it appears that your first order of business is to get the known working plasmid into your L.m. cells. Until you can do the positive control, there is little sense trying to build and work with new ones that you have no confidence in. I would look very carefully at previous work in transforming L.m. and try to duplicate it. Presumably trying to directly duplicate the experiments of the person who developed the working plasmid is the place to begin.

phage434 on Fri Jul 1 00:08:50 2011 said:

I tried this protocol exactly the same for 5 times with the working plasmid. I know there could be some issues with ratio plasmid/bacteria, but I am measuring the concetration of plasmid, giving exactly 1ug as they described, taking care that the volume does not exceed 10% of volumen of bacteria. Also, there could be some issues with preparation of electrocompetent cells, but they have explained really well how to prepare all the media and buffers (to autoclave or not, to filter-sterilize or not, to adjust pH or not) to use.
The only thing that is not so exact as they say is the electroporator. We have this small Biorad electroporator (Micropulser) that has already programs for different bacteria and yeasts and additionally you can change the voltage (I have set it to 1kV), but you cannot change the resistance(it should be 400 ohms) and the capacity (it should be 25uF) and I could not find anywhere in the manual and at the internet the specification for this matter.
Can that really be such a big factor, so I do not observe ANY of transformed bacteria? :/

Make sure you are using the correct width of cuvette. They come in 4mm, 2mm, and 1 mm width, and you need to use the same as your paper does.

Also, pay careful attention to how they prepare the cells. What phase of growth do they harvest at? How are they washed? I would do a viable colony count on the prepared cells both before and after electroporation by plating out serial dilutions on non-selective plates.

How are you recovering the cells? Do you grow them with non-selective medium for an hour or more, followed by plating on selective plates? What do your co-workers do?

Worst case, can you visit the lab that did these experiments and watch how they do it?

phage434 on Fri Jul 1 12:31:52 2011 said:

I am using the 1mm as they do it in this protocol, so it should not be a problem.

phage434 on Fri Jul 1 12:31:52 2011 said:

The protocol for preparing electrocompetent cells is quiet long and tedious. In short: you have to grow O/N culture in BHI, than dilute this suspension to 10% in BHI medium+ 0.5M sucrose (BHIS). Grow till one stage. Add penicillium. Grow till the OD doubles and then cfg and wash with descending volumes of cold sucrose-glycerol wash buffer (SGWB) for several times. add also in one step lysozime. then cfg and wash again with SGWB and cfg and wash with SGWB. aliquot the suspension in 50uL volumes and store at -80 degrees. Everything on ice all the time.

phage434 on Fri Jul 1 12:31:52 2011 said:

I am always doing a negative control (bacteria+ water) and plating on non-selective plates 3rd and 5th dilution and bacteria grow nicely (the plate is full of bacteria, so I never counted the Nu.). But I will try that to see the exact viable colony count. Thank you for this note )

phage434 on Fri Jul 1 12:31:52 2011 said:

I am recovering them as they said in protocol (actually the protocol is very, very detailed for an article with lots of notes, but it still does not work () : with 1mL room temperature BHIS, statically at 30 degrees in incubator for 1,5h. Do the serial dilutions with BHIS and plate them on selective plates with concentration of selective antibiotic as the people from this other lab, from where I have got the plasmid, have recommended to me.
I am the only person in my lab doing cloning stuff, also the youngest one and I have asked the authors quiet a lot of questions already and I really stick to their advices, but still no result. That is why I asked here, if somebody has some extra advice. Thanks for all your comments and notes. It is really good to go with somebody else through the protocol again and check the crucial points of it.

phage434 on Fri Jul 1 12:31:52 2011 said:

Well, maybe I could be able to arrange that on my own cost during my holidays


Thank you once again )

Hi, friend. recently i have done this transformation successfully, i hope the method i show below could help you. You should switch the mode to "Time Constant" , then set the voltage and the time to 1000v and 5ms respectively. I was using the 1 mm cuvette and Biorad electroporator. Good luck for you.

Hi lab_microbe,

 

Did you figure out the problem eventually? I'm now facing the same problem,  with a plasmid  transforming to listeria, I've tried several weeks, couldn't get any colony. I'm exactly following the method described by Monk et al., 2008 (http://aem.asm.org/c...rint/74/13/3921), wondering if anyone has any suggestions.

Thanks.