Recombinant DNA not degraded inside transformed cells, why?

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Holmes
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Recombinant DNA not degraded inside transformed cells, why?

Post by Holmes » Thu Sep 27, 2012 7:38 am

Why is it that recombinant DNA (a foreign DNA) not degraded inside the transformed bacteria?
All bacterial systems have a system to distinguish between the self and non-self DNA. How does recombinant DNA propagate then?

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jonmoulton
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Post by jonmoulton » Thu Sep 27, 2012 4:26 pm

When a foreign DNA enters a bacterial cell, there is a race between methylases and restriction endonucleases. If the DNA is methylated as "self" before the corresponding endonuclease arrives, the DNA will not be sliced.

I'm sure there is more to this story -- help out, anyone?

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Re: Recombinant DNA not degraded inside transformed cells, why?

Post by Holmes » Fri Sep 28, 2012 2:32 pm

Escape from restriction endonucleases is a valid point.
That is totally a chance dependent phenomenon.

Thanks.

Cat
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Post by Cat » Fri Sep 28, 2012 4:21 pm

Yes. The system is not designed to prevent invasion of such magnitude. Also, most lab bacteria have been modified and don't have an active defense system to improve transformation efficiency.

There is another interesting tidbit of information about transformations:

If you transform bacteria with the mixture of two plasmids, you will get some bacteria with one plasmid and some with the other and none with both of them. I have no idea why that would be. If someone knows the answer, please, post.

wbla3335
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Re:

Post by wbla3335 » Fri Sep 28, 2012 6:09 pm

Cat wrote:If you transform bacteria with the mixture of two plasmids, you will get some bacteria with one plasmid and some with the other and none with both of them. I have no idea why that would be. If someone knows the answer, please, post.

I think it's just a numbers game. A very small proportion of cells take up a plasmid during transformation. The probability that one cell will take up both plasmids is thus very, very small.

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Re: Recombinant DNA not degraded inside transformed cells, why?

Post by jonmoulton » Fri Sep 28, 2012 6:18 pm

Here's one from PLOS Genetics today, right on time.

Zhang G, Wang W, Deng A, Sun Z, Zhang Y, et al. (2012) A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria. PLoS Genet 8(9): e1002987. doi:10.1371/journal.pgen.1002987

http://www.plosgenetics.org/article/inf ... en.1002987

Cat
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Re: Re:

Post by Cat » Fri Sep 28, 2012 10:28 pm

wbla3335 wrote:
Cat wrote:If you transform bacteria with the mixture of two plasmids, you will get some bacteria with one plasmid and some with the other and none with both of them. I have no idea why that would be. If someone knows the answer, please, post.

I think it's just a numbers game. A very small proportion of cells take up a plasmid during transformation. The probability that one cell will take up both plasmids is thus very, very small.


Actually, one of my former professors said that it's one plasmid per bacterium. That was years ago. I have not heard anything else on the subject since. I just don't know...

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Post by FlyMusings » Sat Sep 29, 2012 5:56 pm

I think bacteria can take up two plasmids, but taking up plasmids and expressing them is a big toll on the bacteria. So bacteria that took up two plasmids are less healthy than those that took up one plasmid. Over culture, bacteria with one plasmid simply outgrow those with two plasmids.

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Re:

Post by Cat » Sun Sep 30, 2012 3:00 pm

FlyMusings wrote:I think bacteria can take up two plasmids, but taking up plasmids and expressing them is a big toll on the bacteria. So bacteria that took up two plasmids are less healthy than those that took up one plasmid. Over culture, bacteria with one plasmid simply outgrow those with two plasmids.


Nonsense. We routinely transform bacteria with two different plasmids and express both. We just have to do it in steps: 1. transform with plasmid one, 2. make those cells competent, 3. transform with the second plasmid...

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Post by JackBean » Sun Sep 30, 2012 10:05 pm

Yes, bacteria can hold several plasmids. E.g.E. coli Origami cells posses other plasmids used during the protein expression.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: Recombinant DNA not degraded inside transformed cells, why?

Post by Holmes » Wed Oct 03, 2012 7:25 pm

I found some explanation here: (last paragraph)
http://www.tru.ca/faculty/dnelson/cours ... cDNA1.html

Thanks.

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