Low copy plasmid?

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Low copy plasmid?

Postby ChiaraP » Mar 05 2008 6:01 am

Hi!!
Have anyone of you worked with the plasmid pPICZalphaC for the expression of heterologous proteins in Pichia pastoris? I have some troubles in propagating the recombinant vector (the insert of interest cloned into pPICZalphaC) in E. coli strain TOP 10 F'. I obtained some clones and I propageted them in 2 ml of LB low salt medium plus zeocin. The following day, I extracted plasmid DNA using a commercial kit and, after running a sample in agarose gel, I found out that the yield was extremely poor. I made out that it was due not only to a low volume of bacterial culture, but also to the reduced concentration of salt in medium. So, I tried to growth bacteria in 5 ml of "complete" LB medium without antibiotic. I extracted plasmid DNA, but no such luck I did not found any band in the gel. Do you think that pPICZalphaC vector might be a low number plasmid? What's your opinion about it?
Thanks in advance for your attention!!

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Postby mchlbrmn » Mar 05 2008 10:06 am

Do I understand that you grew the plasmid in bacteria without antibiotic? Without antibiotic selection bacteria will loose the plasmid over the course of multiple cell divisions.
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Re: Low copy plasmid?

Postby woodshedder » Mar 05 2008 5:51 pm

ChiaraP wrote:I have some troubles in propagating the recombinant vector (the insert of interest cloned into pPICZalphaC) in E. coli strain TOP 10 F'.


Any particular reason you're using TOP10 F' instead of TOP10? Although I don't think it will matter, since there are no lac promoter/operators on this plasmid (right?).

ChiaraP wrote:I found out that the yield was extremely poor. I made out that it was due not only to a low volume of bacterial culture, but also to the reduced concentration of salt in medium.


Were your overnight cultures turbid or light? The low salt shouldn't matter for E. coli growth. It does matter for Zeocin functionality though.

ChiaraP wrote:So, I tried to growth bacteria in 5 ml of "complete" LB medium without antibiotic. I extracted plasmid DNA, but no such luck I did not found any band in the gel. Do you think that pPICZalphaC vector might be a low number plasmid?

This is a pUC-origin plasmid, it should have a high-copy number. So I reckon even with 1ml of overnight culture, you ought to be able to see plasmid. I agree with mchlbrmn, I wouldn't grow the culture without the Zeocin, but with normal LB, the Zeocin won't work (apparently), so I'd go back to low salt and using Zeocin. If you are growing culture in low salt LB with Zeocin, and you get a turbid overnight culture, then you ought to get a good yield of plasmid. If the cell density in your overnight culture is low, let it grow longer.
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Postby ChiaraP » Mar 06 2008 3:52 am

Hi all!!
Thanks a lot for your replies!!
Well, firstly, I grew my E. coli in LB low salt with zeocin. After an overnight culture at 37°C, I found out that the culture was turbid. Unfortunately, when I extracted plasmid DNA, a faint band was shown on agarose gel. This was the reason why I thought that the poor yield was due to some trouble in the replication of the plasmid DNA. As a consequence, although I know that going without the antibiotic could led to the loss of the plasmid, I tied to grow the clones in LB with the normal concentration of NaCl. After an overnight culture at 37°C, the culture was really turbid. When I exctracted plasmid DNA, I was not able to detect the band on an agarose gel, though.
Concerning the E. coli strain used, TOP 10 F', it was included in the kit that provided Pichia strains and vectors. It is supposed to work well.....
Another concern should be the extraction kit, but it worked well with the extraction of another plasmid DNA, purified at the same time. So, I reckon the poor yield is not due to the kit.
Well, in light of your suggestions, I will come back to low salt LB with zeocin. As 2 ml of bacterial culture did not provide an adequate quantity of DNA, do you think that starting from higher volumes should help?

Thanks a lot for your advices!!
Bye

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Postby kayvee » Mar 06 2008 12:57 pm

I worked with pPICZAlpha A (not C, but don't think that should matter here) and I always used 5mL cultures for plasmid preps. I use Qiagen kit for plasmid prep and I elute in 50ul of TE. My typical concentrations (as determined by UV Abs@260nm) have been 150-200ng/ul. May be it was just that one time you had a problem due to some inexplicable reason. Redo it; you should be okay. One thing I would suggest is to make sure the pH is okay. I had a problem with that once.
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Re: Low copy plasmid?

Postby talkingtree » Jul 03 2009 7:35 pm

ChiaraP wrote:Hi all!!

Thanks a lot for your replies!!

Well, firstly, I grew my E. coli in LB low salt with zeocin. After an overnight culture at 37°C, I found out that the culture was turbid. Unfortunately, when I extracted plasmid DNA, a faint band was shown on agarose gel. This was the reason why I thought that the poor yield was due to some trouble in the replication of the plasmid DNA. As a consequence, although I know that going without the antibiotic could led to the loss of the plasmid, I tied to grow the clones in LB with the normal concentration of NaCl. After an overnight culture at 37°C, the culture was really turbid. When I exctracted plasmid DNA, I was not able to detect the band on an agarose gel, though.

Concerning the E. coli strain used, TOP 10 F', it was included in the kit that provided Pichia strains and vectors. It is supposed to work well.....

Another concern should be the extraction kit, but it worked well with the extraction of another plasmid DNA, purified at the same time. So, I reckon the poor yield is not due to the kit.

Well, in light of your suggestions, I will come back to low salt LB with zeocin. As 2 ml of bacterial culture did not provide an adequate quantity of DNA, do you think that starting from higher volumes should help?



Thanks a lot for your advices!!

Bye



ChiaraP


hi,

it seems like i'm having the same result as you got, i cannot see any bands on agarose gel, i used 4 ml of overnight cultures and i still cant see anything, strange? my strain used was JM109

have you solved the problem by now? ie such as growing in higher volume or others? if so would you like to share :)

by the way have you done any ligations/transformations with your pPic vector? it seems like i cannot propagate at all at 25ug/ml!?!
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Re: Low copy plasmid?

Postby talkingtree » Jul 03 2009 7:40 pm

hi ChiaraP,

so have you solved the problem by now? it seems like i'm having the same problem with JM109 strain growing in 4ml media

also did your propagation worked with your insert? how long does it take to grow such cells? it takes me around 2-3 days to grow up e.coli is this normal?

would be nice if you share your story :D
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Re: Low copy plasmid?

Postby emmafernn » Aug 22 2016 11:37 am

Hello Scientists,

I realise this thread is ancient, but in case someone in the future stumbles across the same problem again...

I was having the exact same problem with pPICZ alphaB in TOP 10 E.coli. Colony PCRs were perfect, but on moving them to overnights - terrible yield.

The solution was to let the colonies grow over TWO nights. And, true to scientific form, I discovered this when I left my overnights on the bench in a rage, and the next day they had turned cloudy ..!

Low salt LB, 37oC, 2 nights. No idea why these bacteria/the plasmids are slow growing/replicating - don't question it. I hope this helps someone in the future :o)

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Re: Low copy plasmid?

Postby arrmcbain » Nov 17 2016 8:31 pm

I'm re-resurrecting this thread in the hope that someone can help.. Does zeocin make E.coli grow very slowly even when they're carrying a zeocin resistance gene? I'm having trouble even getting my pCpG-free Basic plasmid (invivogen) to grow up in the host cells provided with the plasmid on zeo plates provided with the plasmid? I have checked the competency of the cells using a different plasmid and they are fine.. Just not sure why I can't get transformants to grow on my zeo plates..?

[Note in answer to earlier questions on this thread, if your plasmid contains the R6K origin of replication (like mine does - pCpG-free Basic) you need a pir-expressing E.coli host strain such as PIR1 or GT115. If not, well I guess it's just down to culture conditions and a low-copy plasmid..]
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