Is this counter-productive cloning or not? - (Apr/08/2014 )

I spent 1month trying to clone a insert into a vector that we wanted to use for an over-expression study. Basically, it just didn't work. So we tried TOPO and voila.... it worked fine within 1week!

I have now digested the TOPO-vector (carrying our insert) with EcoR1 and got bands as were expected (our insert carries 3 EcoR1-sites within it).

 

Question 1)

But the PCR-produced insert that we cloned into the TOPO-vector is also flanked by EcoR1 on both ends of it, and will after digestion with EcoR1 create 2pieces of 13-18bp fragments (plus the 3 bands created by EcoR1-digestion within the insert itself).

But these 2 fragments don't show on the electrophoresis. Is that because the bands simply are way too small to be visualised?

 

Question 2)

Now I have been told to digest the TOPO-vector (that carries our insert) with restriction-enzymes (for the primers that we used for amplifying the PCR-insert).

This digestion will basically cleave out our insert from the TOPO-vector. Then we will send it all to sequencing.

Isn't it a bit counter-productive to first PCR-amplify a insert, incorporate it into a TOPO-vector and then cleave it OUT AGAIN???!

What is this purpose for?

 

Question 3)

Can you use a TOPO-vector (pCR4) for an over-expression study? If yes, does that mean that I now need to design primers for the promoters that it carries?

1) yes, the 13-18 bp fragments are probably too small or not high enough concentration to see on the gel.

 

2)Yes - normally it is easier to sequence off the plasmid to confirm the insert sequence (longer reads usually)

 

3)Check out the manual for the vector - it should tell you the promoters available (and hopefully give you the sequence and locations if that's what you need).  You shouldn't need to amplify the promoters unless you are wanting to subclone the promoters into another vector.

The plasmid should be used for sequencing. Normally, a sequencing primer will be chosen 5' of the beginning of the insert by 100 bp or so, such that the entire insert can be seen in the sequencing reaction. A second primer,  100 bp 3' of the insert (in the reverse complement orientation) is also often used for a second sequence in the opposite direction. One primer only per sequencing reaction! The pCR4 vector has standard primer  binding sites, M13 forward and M13 reverse, which your sequencing provider almost certainly has primers for, so you can probably avoid adding any primers to the DNA you send them.

 

I don't think pCR4 has vector encoded promoters, so  unless your construct includes promoters, it is unlikely to express (or express well).

Invitrogen also offers expression TOPO vectors. Otherwise you would need to (double)cut and ligate to an expression vector.

PCR4 only has the lac-promoter as far as I can see on the plasmid map. But I guess that can't be used for my over-expression study?

Unless I actually want to fuse my insert to the lac-promoter. So that every time the lac-promoter is expressed, then so is my gene? Sort of like a reporter-system, right?

 

What do you mean with "Otherwise you would need to (double)cut and ligate to an expression vector". I guess you mean to excise the insert out from the TOPO-vector and ligate to an expression-vector, but what is the double meaning?

Also, we tried to do this from the beginning, excise the insert and ligate to a expression-vector, but it NEVER WORKED for 1months time. So that is why we went with TOPO (and it worked). So if I now need to cut it out and fuse to the expression-vector again, isn't that back to step 1 with no results? What was the TOPO-approach for then? Counter-productive it seems.

1 month is nothing unusual for a cloning exercise.  What Trof was referring to is that there are TOPO vectors for expression, not just for cloning.  The otherwise is a return to your original design.

 

Failed cloning into an expression construct could indicate that it is toxic to the cells, you may want to investigate a bacterial line that won't constituitively express the plasmid.

TOPO TA cloning may happen in any direction, it's not specific. If you cut the insert out with a single enzyme, the ligation then will be also direction inspecific. That doesn't matter if you need insert for sequencing only, but actually matters a lot if you have expression vector, which has promoter only on one side. So to ensure directional specificity, you better cut with one enzyme on one side and different on the other side.

Since all TOPO plasmids have multiple cloning site (as other vectors should have too), choosing suitable RE shouldn't be that difficult.

 

Non-expression plasmids may have active bacterial promoter for LacZ but that can't be used in overexpression studies, it's not a strong promoter. Moreover if you want to express in non-bacterial cells you need a specific promoter for that kind of cells (for mammalian cells, CMV promoter is often used, is it's a strong viral promoter).

 

So you said, you cloned your gene into other plasmid (no-expression) sucessfully and then failed in religating it into expression vectors? Or you were just having some old plasmid with insert and you couldn't religate it anywhere else, so you PCRed it out to TOPO?

 

In first case it may be the case of insert toxicity, in the other just failed re-ligation. If it's toxic, TOPO expression vector won't help you, if you can't religate, it may. TOPO just simplifies ligation procedures, but those are not impossible to do, but require more time to optimize, to try over, weeks, months, depending on the difficulty of the insert.. 

 

If your primary goal was to overexpress gene somewhere (or specific mutation of a gene), and that gene is human or from a common organism, probably you should choose different approach from the start. Companies sell already cloned human (probably other too) genes in an ordinary plasmids, and offer simple to transfer from that vector to various types of expression vectors, with many posibilities regarding antibiotic resistance type, C- or N-terminal tags, GFP fusion of co expression.. you name it. There are variable options, Gateway vectors, Origene ORF clones.. they use different systems, depends on you, what you need. You may then play with different expression vectors more easily.

Of course traditional approches works too, but if you unexperienced, they take longer, if you can spend money and don't have time, it's a better solution.

Question 1)

So for 1month I tried to insert my gene into a vector-construct that was given to me by my supervisor. The purpose was to use the final cloned construct for an over expression study.

- I PCR-amplified the insert, flanking it with RE-sites suitable for ligation. Then I digested it to create compatible overhangs, purified it as well.

- The vector itself was also digested to create overhangs (we used same enzyme for the 3' end of both insert and vector, but different enzyme for the 5' but they generated compatible overhangs still).

 

- Up to the ligation, electrophoresis validated the presence and quantity of both insert and vector-DNA. But after the ligation, the ligated DNA-product was all gone.

- At another instance, the ligation went well and electrophoresis validated the prescene and quantity of the final ligated product, but still we got NO GROWTH in our transformed E.coli (we used both XL2 blue and DH5)

 

Would this imply that maybe the insert/vector construct somehow was TOXIC for the cells? And if so, wouldn't my supervisor be aware of that the gene-products would be toxic to the cells?

 

Question 2)

The same cloning, but we used the TOPO-approach and it worked in 3 days and I got colonies. Since we used the same insert (but new vector, pCR4) and we got colonies with the same type of bacteria (DH5).... then now I am thinking that TOXICITY was not a problem in this instance. Right?

We have send the construct for sequencing and awaiting for the data.

It's hardly to say if you supervisor did or didn't knew something. Sometimes protein can be toxic and nobody knew that beforehand.

But using "they should knew about that" as an argument in troubleshooting usually doesn't help at all. You should assume that nothing is validated and check everything again.

 

 

I don't understand much, in what part of transformation did you checked, your plasmid for insert. And how? "Up to the ligation" means what? The ligation reaction is full of vector and insert parts, how exactly did you checked for that?

I don't thing you can validate anything before you got first colonies. And after that, colony PCR often gets false positive (or at least false slightly positive).

 

The toxic product in bateria is negatively selected out, on the other hand, bacteria needs the abtibiotic resistance gene to survive, so quite othen in such case they recombinate and "cut out" already ligated plasmid. Some evidence in this can be seen, if the site of the insert (now missing) is either mutated different ways in different plasmids, or there actually is a part of the insert, but not the whole one.

 

As said before, pCR4 is not an expression plasmid, if it doesn't express the product, bacteria have no need to cut it out. 

 

 

Still there are two options, first you just failed with ligation. In the first case the vector recirculated and your thest were false positive or just done it at wrong time with a wrong method. Therefore TOPO ligation was succesfull since is more efficient and vector doesn't re-ligate .

In the second, you managed to ligate well, but indeed the priduct is toxic when expressed (that doesn't happen in PCR4). You may test this if you are able to religate to a different non-expression plasmid sucessfully. But, succesfull ligation in such case may still not exclude that the other ligations were failed.

But I'm not sure how you make it grow in any expression plasmid, what kind of cells you need to use (and of course I don't know what promoter are you using, CMV promoters for example in theory should not be active in bacterial cells, but they still are a bit, much less than in mammalian cells, but still).