In this question, determine the values for the linking number (L), super helical turns (AL), the twisting number (T) and the writhing number (W = supercoiling number) initially and after Steps 1 -4. Each part of your answer will be for the plasmid as a whole. It will be helpful to keep track of whether a nick is present or not. Below the table, explain your answers. Assumptions: Choose a reference state as explained in class. Once you have chosen this reference state at the outset of the problem, do not change it at any time throughout the problem. (After all, what good is a reference state if it is not constant?) Throughout this problem, assume that all duplex DNA is B-DNA (10 bp/turn), except where it is mentioned otherwise. Initial State: A 5000 bp plasmid is completely in the B-DNA conformation under normal conditions and has 25 underwindings (or 25 negative superhelical turns). Step 1: The plasmid is nicked once in one strand and continues to be B-DNA. Step 2: There is a region of 240 bp in the plasmid that is normally in the B-DNA conformation, butit can be induced to adopt the A-DNA conformation by changing salt concentration. The plasmid DNA is manipulated such that this B-DNA to A-DNA transition occurs in the 240bp region. The rest of the plasmid remains B-DNA. Important Hints: 1.A-DNA was NƠT discussed in class. Assume: right-handed, 12 bp/turn. After step 2, the plasmid is “relaxed", since it remains nicked. HOWEVER, it is not in the same relaxed state as the reference state. In spite of the change in salt concentration, do not change the reference state. Step 3: The plasmid is re-ligated (i.e., the nick is re-sealed). You will have to think through whether DNA Ligase changes DNA topology (is it a topological enzyme?), by thinking about the reaction catalyzed by DNA ligase. Step 4: The plasmid is returned to the original salt concentration, and all of the plasmid regains the original B-DNA conformation. (Note: no nicking occurs in Step 4.) Initial Step 1 Step 2 Step 3 Step 4 Step 5 L Delta L T W Nick? No Yes Yes No No You Decide
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Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
In this question, determine the values for the linking number (L), super helical turns (AL), the twisting
number (T) and the writhing number (W = supercoiling number) initially and after Steps 1 -4. Each part of your answer will be for the plasmid as a whole. It will be helpful to keep track of whether a nick is
present or not.
Below the table, explain your answers.
Assumptions:
- Choose a reference state as explained in class. Once you have chosen this reference state at the
outset of the problem, do not change it at any time throughout the problem. (After all, what good is a
reference state if it is not constant?)
- Throughout this problem, assume that all duplex DNA is B-DNA (10 bp/turn), except where it
is mentioned otherwise.
Initial State: A 5000 bp plasmid is completely in the B-DNA conformation under normal conditions and
has 25 underwindings (or 25 negative superhelical turns).
Step 1: The plasmid is nicked once in one strand and continues to be B-DNA.
Step 2: There is a region of 240 bp in the plasmid that is normally in the B-DNA conformation, butit can
be induced to adopt the A-DNA conformation by changing salt concentration. The plasmid DNA is
manipulated such that this B-DNA to A-DNA transition occurs in the 240bp region. The rest of the
plasmid remains B-DNA.
Important Hints:
1.A-DNA was NƠT discussed in class. Assume: right-handed, 12 bp/turn.
- After step 2, the plasmid is “relaxed", since it remains nicked. HOWEVER, it is
not in the same relaxed state as the reference state. In spite of the change in salt concentration, do not
change the reference state.
Step 3: The plasmid is re-ligated (i.e., the nick is re-sealed). You will have to think through whether
DNA Ligase changes DNA topology (is it a topological enzyme?), by thinking about the reaction
catalyzed by DNA ligase.
Step 4: The plasmid is returned to the original salt concentration, and all of the plasmid regains the
original B-DNA conformation. (Note: no nicking occurs in Step 4.)
| Initial | Step 1 | Step 2 | Step 3 | Step 4 | Step 5 | |
| L | ||||||
| Delta L | ||||||
| T | ||||||
| W | ||||||
| Nick? | No | Yes | Yes | No | No | You Decide |
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