Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Superdupercoiling: what affects the extent of supercoiling?

supercoil supercoiling plasmid

  • Please log in to reply
10 replies to this topic

#1 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 26 August 2014 - 08:09 AM

What factors affect plasmid DNA supercoiling? I know already that a nicked plasmid loses its supercoiling but what else?

 

Is the same plasmid produced in a different bacterial strain or species supercoiled to the same extent? Does the sequence of the plasmid have any effect?



#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,805 posts
133
Excellent

Posted 27 August 2014 - 03:44 AM

you can start with these wikipedia pages/sections on conformation of plasmids and supercoiling then look at some of the references listed.


Edited by mdfenko, 27 August 2014 - 03:45 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#3 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 28 August 2014 - 10:54 AM

Maybe I should have written my question differently (I've already perused Wikipedia and it contains no information that I'm interested in that I didn't already know from elsewhere). I'm most interested in what I can do to increase the quality of my mini- and maxi-prep plasmids for subsequent transfection by lipofection and electroporation and right now I'm looking into supercoiling. I already take care not to vortex or repeatedly freeze-thaw. I think the pH and ionic conditions are outside of my control and trust Lonza and Life Technologies to take care of that aspect in the development of their reagents!



#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 28 August 2014 - 11:18 AM

If you have covalently closed DNA (un-nicked plasmid) then treatment with either Topoisomerase I or DNA Gyrase will either remove or add supercoiling, respectively. See the reagent descriptions at NEB.COM.



#5 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 28 August 2014 - 12:39 PM

Thanks! The NEB doesn't mention transfection which suggests that my idea might not be that smart. I've sent an e-mail to Lonza asking if the Gyrase could be used in this way. A topoisomerase would relax the plasmid when it hits the nucleus, right?


Edited by seanspotatobusiness, 28 August 2014 - 12:40 PM.


#6 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,805 posts
133
Excellent

Posted 29 August 2014 - 03:27 AM

A topoisomerase would relax the plasmid when it hits the nucleus, right?

no nucleus in bacteria.


talent does what it can
genius does what it must
i do what i get paid to do

#7 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 30 August 2014 - 01:44 PM

 

A topoisomerase would relax the plasmid when it hits the nucleus, right?

no nucleus in bacteria.

 

 

I know but I'm transfecting animal cells.



#8 Bio-Lad

Bio-Lad

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 85 posts
16
Good

Posted 06 September 2014 - 06:21 PM

Treating plasmids with gyrase for transformation into bacteria can increase efficiency by a lot (I've read up to 9-fold for large plasmids).  There is an old nucleic acids research paper (is 1995 really that old, though?) where the authors did this (see "DNA gyrase improves DNA transformation of E.coli cells with large recombinant plasmids", I can send the PDF if you need it.)  I know that paper is on transformation and not transfection but there are other studies that show transfection is increased as well (see "Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography.") so I would think your idea merits trying, even if just to tell us if it worked biggrin.png .


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#9 Bio-Lad

Bio-Lad

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 85 posts
16
Good

Posted 06 September 2014 - 06:34 PM

I also saw mention somewhere (I'd have to find it again) about increased yield of supercoiled plasmid from e. coli over-expressing gyrase to give better transfection rates (although I believe that was in the context of DNA vaccines, not lipofection).


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#10 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 08 September 2014 - 06:18 AM

Hey Bio-Lad,

 

I'm still looking into this. I've seen a few of the same articles you have.

 

Lonza wasn't that helpful. I think he was saying not to bother. I've included the response below.

 

I couldn't find the paper discussing improved supercoiled yields from gyrase over-expression. I only found this one that said there were reduced overall yields: http://www.ncbi.nlm....les/PMC2735187/

 

The Life Technologies ChargeSwitch kit claims to give almost entirely supercoiled plasmid on this page:

HdkO0iW.png

However on this page they claim that their kit results in identical (not superior) transfection efficiency to their regular anion-exchange-based kit:

LMpVzO0.png

 

 

 

Lonza e-mail

 

Thank you for your email. Indeed DNA quality plays a major role in transfection efficiency and also viability.

In principal nicked plasmid DNA (which is also called open circle; oc) seemed to be express less when transfected in a cell. This might be due to the fact that DNA is faster degraded when entering the cytoplasm and/ or nucleus. So it is best to have an endotoxin-free plasmid purification which showed most of the plasmid in its supercoiled form.

However due to the intracellular topoisomerases I and II the plasmid will be immediately relaxed when delivered to the cell . So, an incubation with Gyrase will change the supercoiling of the plasmid (visible on an agarose gel)  but by transfecting it into the cell it will be relaxed by the intracellular topoisomerase enzymes.

Please find here some more information about the importance of supercoil for plasmid preparations and transfection into mammalian cells:

 

Cytotechnology. Jan 2011; 63(1): 7–12.

Published online Dec 1, 2010. doi:  10.1007/s10616-010-9322-9

PMCID: PMC3021145

Separation of supercoiled from open circular forms of plasmid DNA, and biological activity detection

Huangjin Li, Huaben Bo, Jinquan Wang, Hongwei Shao, and Shulin Huang

 

I hope my answers are of any help for you. Please let me know if you have further questions.

Best wishes,

 

Elke



#11 seanspotatobusiness

seanspotatobusiness

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 175 posts
7
Neutral

Posted 11 September 2014 - 07:07 AM

I found an article which says that supercoiling has no effect on the efficiency of lipofection - they test five different reagents and five different cell lines. They don't mention electroporation though; that could make a difference?







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.