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ccdb toxin


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15 replies to this topic

#1 pito

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Posted 30 July 2014 - 12:33 AM

hallo all,

 

just curious: anyone here using this toxin as a selection marker in the gateway system? I have been using it lately but to my surprise I noticed that it is not that effective. I still get lots of cells that harbor the plasmid with this toxin.

 

I am wondering others might have seen this too? 

 

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#2 phage434

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Posted 30 July 2014 - 05:06 AM

Yes, I have a lot of experience with this. The problem is that mutations will occur in the fragment, and they are (of course) selected for. Evolution really wants these cells to survive. We switched away from using it, moving to an RFP cassette, whcih can be used to easily identify parent plasmid. But the thing that really worked for us was to start PCR amplifying the plasmid backbone with minimal template, then DpnI digesting the product, practically eliminating parent plasmid. That, and using a cloning plasmid with diferent antibiotic resistance than the source of the inserts.

Shetty R, Lizarazo M, Rettberg R, Knight TF. Assembly of BioBrick standard
biological parts using three antibiotic assembly. Methods Enzymol.
2011;498:311-26. doi: 10.1016/B978-0-12-385120-8.00013-9. PubMed PMID: 21601683.


#3 pito

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Posted 30 July 2014 - 08:40 AM

Yes, I can see your point.

According to the seller of the system it should not be such a problem. I just noticed it the last few weeks that the system is not that bulletproof!

I also sequenced the gene itself and it still is the original fragment! Perhaps in some colonies it is indeed a problem with mutations.

 

Your system, I remember it from a while ago, seems nice, but for the work I am doing its too much work I am afraid.

 

And the biggest problem is pretty much the fact that the genes I have to use are given to me in a gateway plasmid.

So I am pretty much forced to use it.

 

 

The PCR amplification and digesting , restriction free cloning as I know it, is indeed a very usefull system. I pretty much use it all the time when I can!

Makes me wonder why so many people still keep getting stuck working with restriction enzymes and ligations and so on.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#4 BioMiha

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Posted 06 August 2014 - 11:31 AM

What kind of competent cells did you use? Bugs harbouring the F'-episome survive ccdB selection, e.g. XL1 Blue and derivatives. You should check the genotype of the bugs before giving up on the system altogether biggrin.png



#5 pito

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Posted 06 August 2014 - 09:29 PM

I am using top10 F' cells from life technologies.. They do contain a part of the F plasmid, but not the ccdB gene (at least it should not contain it!).

 

What kind of competent cells did you use? Bugs harbouring the F'-episome survive ccdB selection, e.g. XL1 Blue and derivatives. You should check the genotype of the bugs before giving up on the system altogether biggrin.png

 


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#6 BioMiha

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Posted 07 August 2014 - 02:13 AM

F' is a huge plasmid and all of its genes are rarely described in the manuals. Essentially any DH5-alpha strain (e.g. OneShot TOP10) should work as long as it doesn't have the F' episome.



#7 phage434

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Posted 07 August 2014 - 06:53 AM

I've never tried ccdB with an F' strain. I avoid those strains, since most have additional resistance genes that get in the way of routine cloning.



#8 pito

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Posted 07 August 2014 - 08:31 AM

F' is a huge plasmid and all of its genes are rarely described in the manuals. Essentially any DH5-alpha strain (e.g. OneShot TOP10) should work as long as it doesn't have the F' episome.

The cells I have , top10 F' cells, they only contain a short piece of the F plasmid! It should be fine for ccdb selection (its even mentioned in the website (life technologies) that they are not ment for ccdb propagation.

 

I , personally, would have used the regular top10 cells, not the top10F cells, but for some obscure reason (we don't even work with phages or whatever reason you might need the F episome) they got the Top10F ones...


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#9 pito

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Posted 07 August 2014 - 08:32 AM

I've never tried ccdB with an F' strain. I avoid those strains, since most have additional resistance genes that get in the way of routine cloning.

Tetracycline is indeed coded on that F' episome, but for me its not a problem, but you are right, I would avoid the F' ones too, but I have to work with I have been given at the moment.

In the future I might change to a different strain.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 BioMiha

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Posted 07 August 2014 - 08:35 AM

ccdB is the toxin and ccdA is the antitoxin. So if ccdA is present on the F' episome they won't be selected against even if they haven't taken up your insert. That's why you see background.



#11 pito

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Posted 07 August 2014 - 08:40 AM

Thats what I ment..

I know how the gateway system works...

 

The ccdA should not be present on that particular F plasmid...

The F plasmid is almost never complete when used in cloning (in strains)


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#12 pito

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Posted 11 August 2014 - 02:00 AM

I've never tried ccdB with an F' strain. I avoid those strains, since most have additional resistance genes that get in the way of routine cloning.

You are right.

I just do not buy them because they cost more and I do not need the F episome.

And see below.

 

 

ccdB is the toxin and ccdA is the antitoxin. So if ccdA is present on the F' episome they won't be selected against even if they haven't taken up your insert. That's why you see background.

Its good that I am a bit picky and always checking things rather than just depending on others all the time.

It turns out they do contain this antitoxin! Pretty weird life science says on their website its not for ccdb propagation... I assumed they removed it from the F episome (since its not the complete one... but they give hardly any details about it).

 

So you are right, its not good to use them!

Pretty funny, they have been using them for a while...good that I checked everything.

Will save me a lot of fuzz.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#13 BioMiha

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Posted 11 August 2014 - 02:39 AM

Glad to hear you've managed to make sense of it all. The F' episome is so big that they rarely write it all down, so even if the gene you're looking for is not specifically written, it might still be there. I doubt that they've re-engineered the episome, I reckon it's all there they just don't tell you.



#14 pito

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Posted 11 August 2014 - 03:17 AM

Glad to hear you've managed to make sense of it all. The F' episome is so big that they rarely write it all down, so even if the gene you're looking for is not specifically written, it might still be there. I doubt that they've re-engineered the episome, I reckon it's all there they just don't tell you.

yeah, but they should not mention its not for ccdb propagation... 

Ok granted: the antitoxin is not 100% , but stil.. do not mention it on your website.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#15 Bio-Lad

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Posted 14 August 2014 - 07:13 AM

I realize I'm late to this party but FYI, Top10 F' cells do contain the CcdB operon and have been used in studies for propagation and analysis of ccdB (Ex: http://mbu.iisc.erne...vgrp/PDF/80.pdf).  IIRC, the reason they are not recommended for propagation of CcdB plasmids is that while CcdA allows for survival in the presence of low levels of CcdB, the lack of the gyrase gyrA462 mutation means that if levels get high enough (say from a CcdB selection-based cloning vector), they will still die.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/





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