Recombinant DNA not degraded inside transformed cells, why?

[Holmes]

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?

[jonmoulton]

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?

[Holmes]

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

Thanks.

[Cat]

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]

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.

[jonmoulton]

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]

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...

[FlyMusings]

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.

[Cat]

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...

[JackBean]

Yes, bacteria can hold several plasmids. E.g.E. coli Origami cells posses other plasmids used during the protein expression.

[Holmes]

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

Thanks.