Restriction Mapping of Plasmid Assignment

Question

pUWL22 is a circular, double stranded, 10.5 kb plasmid. It contains a gene encoding an enzyme that confers ampicillin resistance in the host bacterium. Cloning into the kpn I and Sst I sites abolishes ampicillin resistance, whereas cloning into other sites on the plasmid does not. Digestion with the following restriction enzymes singly and combination results in the following data:

Enzyme Digest     ()()()()()()   Fragments Produced (kb)

Hind III  ___________________________10.5

Kpn I  _______________________________10.5

Not I  _______________________________10.5

Pvu II  ______________________________10.5

Sst I  _______________________________10.5

Stu I  _______________________________10.5

Hind III + Kpn II  ___________________8.5  _______________2.0

Hind III + Not I _________________8.0  __________________2.5

Hind III + Pvu II ________________6.0  __________________4.5

Hind III + Sst I _________________7.5  __________________3.0

Hind III + Stu I _________________7.5  __________________3.0

Stu I + Not I  ____________________10.0  __________________0.5

Stu I + Pvu II  ____________________7.5  __________________3.0

Now the actual questions:

A) Create a circular map of pUWL22, designating the Hind III site as the position 0 on the map. Once you have completed the map, indicate the relative position of the ampicillin gene.

My plasmid mapping:

What I need help with in indicating the relative position of the ampicillin gene. In the statement above it said "Cloning into the Kpn I and Sst I sites abolishes ampicillin resistance." Does that mean its inbetween Sst 7.5 and Kpn 8.5? Please tell me where it is and why its there.

P.S. I made a mistake on the on the Sst I enzyme, it's suppose to be @ 7.5 not 7.8.

The question after that is:

B) Propose 2 other double digests that could be done to confirm your analysis. Predict the sizes of the fragments that would be generated from these digests.

Please answer this and explain why.

Answer 1

A) Here is the correct map:

You made a mistake on your map at the PvuII site (it is not on 6.5kB from the start of the plasmid, but on 6kB).

Can the Kpn I not go on the 8.5 site, it still creates the 2 and 8.5, so isn't there more than 1 correct option for plasmid map?

Yes. What you need to do in order to make the correct map is try all possible positions of the restriction sites:

1. You start with HindIII and place it at position 0 of your vector.
2. The KpnII site can be either at 2kb (left of HindIII) or at 8.5kb (right of HindIII). Draw both versions of the plasmid.
3. You place the NotI site that can either be at 2.5kb or at 8kb. Now you have four different possibilities for the plasmid:
   1. HindIII[0], KpnII2, NotI[2.5]
   2. HindIII[0], KpnII[8.5], NotI[2.5]
   3. HindIII[0], KpnII2, NotI[8]
   4. HindII[0], KpnII[8.5], NotI[8]
4. Then you do the same for the rest of the restriction sites. When you get to the last two digests, there is only one option for the correct plasmid left in the following order, depicted on the plasmid map below.

I have to point out though that StuI and SstI can both be at 3kb (they can be next to each other). Then the 2 fragments from the digest will be ~3kB and ~7.5kB. So technically, there are two possible versions: correct

a) HindIII[0], KpnII2, NotI[2.5], StuI[3], PvuII[6], SstI[7.5]

b) HindIII[0], KpnII2, NotI[2.5], StuI[3], SstI[3] PvuII[6]

If u can, can u please elaborate on question b as to why there is one correct option for the plasmid map? Also, what do they mean by double digest to confirm ur analysis?

A double digest means that you cut your plasmid with two different restriction enzymes at the same time. As you already have a putative map of the plasmid, you can predict which would be the expected bands. If you observe the expected sizes, then your design is correct and your analysis are confirmed.

To check which one is correct I would do a double digest with: 1) SstI and PvuII, generating a 1.5kb and a 9kb bands if design a) is correct or 3kb and a 7.5kb bands if design b) is correct 2) StuI and SstI, generating a 4.5kb and a 6kb bands if design a) is correct or a 10.5kB band if design b) is correct

To confirm the "ampicillin gene" starts at kpn I 2.0 and end at the 3.0 stu I site right?? So the amp gene is 1 kB long?? So the relative position of the amp gene starts at 2.0 and ends at 3.0. Why is it that in the question it says cloning into the kpn I and sst I sites abolishes ampicillin resistance whereas cloning on other sites does not? What do they mean by this? Is the ampicillin resistance gene diff from the ampicillin gene??

The ampicillin resistance gene and the ampicillin gene are the same. It refers to an enzyme (beta lactamase) which degrades ampicillin, and thus the cells that harbour the ampicillin-containing plasmid can survive when plated on ampicillin.

Are you sure that the resistance is disrupted if cloning into SstI site and not StuI? If this is really the case, then the StuI site and SstI sites are next to each other, as first you have the SstI site, followed by StuI. The amp gene The gene is a little above 1kb, so it starts upstream of KpnII and ends right after SstI, but it doesn't disrupt StuI. If something is cloned into KpnII or SstI sites, then the open reading frame of amp is disrupted, and the protein cannot be functional.

Answer 2

Heres the solution

i got 1 mark off for the position of Not I