Cloning Large Fragments

[apricimo]

Hello,

 

I am cloning a large, 10 kb, DNA fragment and I was wondering if anyone has had any experience with cloning large chunks of DNA.

 

Background: The 10 kb DNA fragment has four genes in an operon. I would like to basically take this operon and express the proteins in E. coli so that they can do their thing (i.e. make the product that they make).

 

Anyway, I heard different things from different people as far as what subcloning vectors to use or not use. For example, pGEM-T some people say use some people say that my insert is too large. TOPO vector I understand will work for something like this.

 

 

My questions:

 

1.) What subcloning vector should I use for a fragment this large?

2.) What expression vector should I use to express my protein in E. coli with a fragment this large.

 

 

The proteins are presumable cytosolic and come from a gram positive bacteria if that makes any difference in terms of expression vectors. I should make it clear that I am not trying to purify the proteins in the end rather I am trying to isolate the product that they make.

 

Any help would be awesome. Thanks in advance

Edited March 2, 2011 by apricimo

[CharonY]

Generally the cloning efficiency is reduced with fragment size, and transformation efficiency is inversely correlated with overall vector size. Both vectors are relatively small, so the overall construct should work out.

There are other vector systems designed for larger fragments (e.g. comsids or BACs) but for 10 kb it is a bit of an overkill. It may require some tries, but I do not see any critical problems.

 

 

Functional heterologous expression is tricky, though. You need the right promotor and then the products have to be functional thing in the new organism.

In order for that to work you would need either include the native promotor and hope that it will work in E. coli, which is rather unlikely, considering that it comes from a Gram+ bacterium.

The alternative is to get a inducible plasmid with a compatible promotor and only insert the promoterless operon. Traditional expression vectors are used for overexpression, however, and this may not be well suited (in the worst case it will kill of the bacterium). I would search for some inducible, low level promotors as a first starting point (I do not remember any off the top of my head, though).

[apricimo]

Generally the cloning efficiency is reduced with fragment size, and transformation efficiency is inversely correlated with overall vector size. Both vectors are relatively small, so the overall construct should work out.

There are other vector systems designed for larger fragments (e.g. comsids or BACs) but for 10 kb it is a bit of an overkill. It may require some tries, but I do not see any critical problems.

 

 

Functional heterologous expression is tricky, though. You need the right promotor and then the products have to be functional thing in the new organism.

In order for that to work you would need either include the native promotor and hope that it will work in E. coli, which is rather unlikely, considering that it comes from a Gram+ bacterium.

The alternative is to get a inducible plasmid with a compatible promotor and only insert the promoterless operon. Traditional expression vectors are used for overexpression, however, and this may not be well suited (in the worst case it will kill of the bacterium). I would search for some inducible, low level promotors as a first starting point (I do not remember any off the top of my head, though).

 

I heard about a "rule of thumb" that you need your vector to be twice the size of the insert. So for a 10 kb fragment I would need a vector that is 20 kb. I can't think of any vectors like this and have you or anyone heard of this rule of thumb?

[CharonY]

It helps with regards to the efficiency of ligation (as religation would occur more often than ligation with the large fragment). Especially with blunt end ligation it will be tricky. However, at a certain size the overall cloning efficiency goes down, so that it does not help anymore. It is more time efficient to repeat the cloning experiment than trying to create a 30 kb plasmid. It depends on the backbone a little bit, too. But 10 kb should still work (albeit with low efficiency). Over 15 kb I would look for a different vector system.

[apricimo]

It helps with regards to the efficiency of ligation (as religation would occur more often than ligation with the large fragment). Especially with blunt end ligation it will be tricky. However, at a certain size the overall cloning efficiency goes down, so that it does not help anymore. It is more time efficient to repeat the cloning experiment than trying to create a 30 kb plasmid. It depends on the backbone a little bit, too. But 10 kb should still work (albeit with low efficiency). Over 15 kb I would look for a different vector system.

 

excellent