[madscientist]

Hi, I have a question im trying to answer which ive had a decent attempt at, but am unsure if im correct or not. The question reads as follows:

Describe how you could clone the following three fragments into the pBR322 plasmid, and how you would select bacterial colonies containing your recombinant plasmids. If you think it is not possible to clone the fragment , explain why.

a. A 2.5 kb PstI fragment
b. A 1.8 kb EcoRI-BamHI fragment
c. A 2.1 kb PstI-BamHI fragment

My working so far is as follows:

a.) A 2.5 kb PstI fragment
step 1:
 2.5kb PstI fragment carrying a gene, ends of fragment formed by digestion with PstI

Step 2:
Cut the pBR322 plasmid with PstI. The ampicillin resistance gene is cut into two peices and is therefore inactive (ampicillin sensitive)

step 3:
Ligate the 2.5 kb PstI fragment to the linearised plasmid.

step 4:
Transform the ligation mixture into bacteria (E. coli cell). The recombinant plasmid has an intact tetracycline resistance gene, so the bacteria can be selected by growth on plates containing tetracycline. The recombinant plasmid is ampicillin sensitive. By using replica plating which involves transfering colonies to duplicate plates, one containing ampicillin and the other tetracycline, the recombinant plasmid will be the colony that grows on the tetracycline plate but dies on the ampicillin plate.

b. A 1.8 kb EcoRI-BamHI fragment
Not sure how to answer this one, all i can think of is that its not possible because the recombinant plasmid would still have an intact tetracycline and ampicillin resistance gene. Therefore selection of bacterial colonies containing the recombinant plasmids would be impossible.

c. A 2.1 kb PstI-BamHI fragment
Not sure about this one either, Im guessing its not possible due to the recombinant plasmid not having an origin of replication (ori) and therefore impossible to clone.

Am i even close to being right?

I know its a long, drawn out and complicated question but any help at all would be appreciated.

Cheers,

madscientist

[Yggdrasil]

a.) A 2.5 kb PstI fragment
step 1:
 2.5kb PstI fragment carrying a gene, ends of fragment formed by digestion with PstI

Step 2:
Cut the pBR322 plasmid with PstI. The ampicillin resistance gene is cut into two peices and is therefore inactive (ampicillin sensitive)

step 3:
Ligate the 2.5 kb PstI fragment to the linearised plasmid.

step 4:
Transform the ligation mixture into bacteria (E. coli cell). The recombinant plasmid has an intact tetracycline resistance gene, so the bacteria can be selected by growth on plates containing tetracycline. The recombinant plasmid is ampicillin sensitive. By using replica plating which involves transfering colonies to duplicate plates, one containing ampicillin and the other tetracycline, the recombinant plasmid will be the colony that grows on the tetracycline plate but dies on the ampicillin plate.

Good answer.  Alternatively, you could treat the cut vector with alkaline phosphatase to prevent religation of the vector.  This eliminates the need to replica plate.

b. A 1.8 kb EcoRI-BamHI fragment
Not sure how to answer this one, all i can think of is that its not possible because the recombinant plasmid would still have an intact tetracycline and ampicillin resistance gene. Therefore selection of bacterial colonies containing the recombinant plasmids would be impossible.

1) Isn't the BamHI site in the tet^r gene?
2) When you perform your ligation, what products would you expect?  Would you need to select against any plasmids which do not have the insert?

c. A 2.1 kb PstI-BamHI fragment
Not sure about this one either, Im guessing its not possible due to the recombinant plasmid not having an origin of replication (ori) and therefore impossible to clone.

The same as above, but:
3) Would the resulting plasmid be very useful?

For reference, the map of the plasmid can be found here:
http://www.fermentas.com/techinfo/nucleicacids/mappbr322.htm

[edit: changed a lot of the answers]

[madscientist]

1) Isn't the BamHI site in the tetr gene?

Ok so if the BamHI end of the fragment cuts the amp^R gene in pBR322, what does the EcoRI end cut?

Im realy lost with this , after cutting the plasmid with the EcoRI-BamHI fragment would the linear molecule look like this? (see attachment, GATC and CTAG = BamHI)

if this is how the cut plasmid looks, where does the BamHI end of the fragment join or ligate to?

[madscientist]

Am I over complicating it? is it just that if the plasmid is cut with a two-peice fragment like the EcoRI-BamHI fragment that a segment is cleaved out of the plasmid, hence leaving a linear plasmid with BamHI at one end and EcoRI at the other? see attachment, AATT = EcoRI and CTAG = BamHI

[Yggdrasil]

The diagram in the second pdf looks more right than the diagram in the first pdf.  But, according to the site I posted, the EcoRI site is between the amp^r and tet^r genes.  So, you will get two fragments:  One small fragment will contain some unimportant sequence and part of the tet^r gene.  The larger fragment will contain the entire amp^r gene, the entire origin of replication, the entire rop gene, and part of the tet^r gene.  If your insert ligates with the smaller fragment, then it will produce a circular plasmid with no origin of replication which will not propagate after you transform it into bacteria.  However, if your insert ligates with the larger fragment, then you will get a plasmid with your insert, the entire amp^r gene, the entire origin of replication, and the entire rop gene, which will be a viable plasmid. 

So, in order to clone the  EcoRI-BamHI fragment, you would want to digest the vector with EcoRI and BamHI, then run the digestion on a gel.  This will separate the larger fragment from the smaller fragment.  You can cut out the band containing the larger fragment, purify it from the gel, and then use that as the starting material in your ligation.

(btw, I am typing this while taking a break from doing some cloning of my own in lab)

[madscientist]

I think ive had a lightbulb moment, if an EcoRI-BamHI fragment was cloned into pBR322 the following would happen:

Step 1:
Cut pBR322 with the EcoRI-BamHI fragment, the ecori end cuts the plasmid at the position in between the tetR and ampR, and the BamHI end cuts the plasmid at the ampR site

Step 2:
Ligating or attaching the EcoRI-BamHI fragment to the linear molecule, the BamHI end bonds to the complementary BamHI sequence on the plasmid and the EcoRI end bonds to its complementary sequence on the plasmid

Therefore you end up back where you started, no change between the plasmid you started with and the recombinant plasmid.

Is this right?

madscientist

[Yggdrasil]

I think ive had a lightbulb moment, if an EcoRI-BamHI fragment was cloned into pBR322 the following would happen:

Step 1:
Cut pBR322 with the EcoRI-BamHI fragment, the ecori end cuts the plasmid at the position in between the tetR and ampR, and the BamHI end cuts the plasmid at the ampR site

Step 2:
Ligating or attaching the EcoRI-BamHI fragment to the linear molecule, the BamHI end bonds to the complementary BamHI sequence on the plasmid and the EcoRI end bonds to its complementary sequence on the plasmid

Therefore you end up back where you started, no change between the plasmid you started with and the recombinant plasmid.

Is this right?

madscientist

Yes, if you do not separate the two fragment that result from the digestion of pBR322 by EcoRI and BamHI, then the two fragments can religate and form the orginal plasmid again.  However, if you separate the two fragments, and ligate in the presence of a different fragment, you can in essence replace one of the fragments with a different fragment.  See attached image.

[madscientist]

I can't thank you enough for your help Yggdrasil, although the process looks easy in hindsight I wouldn't have been able to get my head around it without your assistance.

Much appreciated,

madscientist

btw, where did you get that cloning.PNG pic from?

[Yggdrasil]

I made it myself on powerpoint/MS paint.  Powerpoint is a pretty good way to make figures.

[madscientist]

In the case of part c, where there is a 2.1kb PstI-BamHI fragment to be cloned into the plasmid pBR322, I know the following would happen.



But how would you separate the resulting two linear molecules to prevent religation?

cheers,

madscientist

P.S  I understand that if you were able to avoid religation by separation, that one of the recombinant plasmids would not have an origin of replication and therefore will not propogate...., and the other would be sensitive to ampicillin and tetracycline, therefore not possible to grow and select colonies.... is this right?

[Yggdrasil]

You would perform a gel extraction (provided that the lengths of the two fragments are different enough).

Also, you are absolutely correct that the recombinant plasmid would have no fucntional antibiotic resistance genes.

[madscientist]

The same as above, but:
3) Would the resulting plasmid be very useful?

Last question I swear, even if the recombinant plasmid from the last example has no functional antibiotic resistance genes, would it still be possible to clone it? i.e growing it on plates with no antibiotic, you could then select colonies of the recombinant plasmid using replica plating as shown below could'nt you?

[madscientist]

That image didnt turn out to well so here it is again.

[Yggdrasil]

How would you separate the bacteria with your plasmid from bacteria without your plasmid?

[madscientist]

Once you know which colony it is on plate III (no antibiotic) couldnt you just swab it and grow that colony on another plate that contains no antibiotic?