Indicate what enzyme you would add to ligate the cut vector and protein Y insert together

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State whether the cloning is directional or not and explain why

 

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Write out the answer to the question below on a separate sheet(s) of paper

1. You wish to create a clone that will express protein Y with an N-terminal His tag i.e. 6 histidine amino acids in a row. (Proteins with this tag can be bound to a resin through the tag, purified away from contaminating proteins by washing, and then eluted off the resin.) You have a vector that has the coding sequence for a 6 X His tag followed by a multiple cloning site (MCS).

The partial sequence of this vector is given below:

5

 

The partial sequence of the cDNA for protein Y is given below:

6

 

The cutting sites for selected restriction enzymes are given below:

7

 

 

 

 

Your job is to design a restriction cloning scheme to insert the cDNA for protein Y into the MCS of the vector, so that a 6 X His – protein Y fusion protein can be expressed. (The frame of cDNA for protein Y must be maintained). The ONLY reagents available to you are the restriction enzymes BamH I, Hind III, Nsi I, Pst I, Sal I, and CIP (calf intestinal phosphatase), T4 DNA ligase, and the appropriate buffers for each enzyme.

 

Criteria to consider:

a) It does not matter how many amino acids are present between the His tag and protein Y components.

b) The protein Y component can start no later than amino acid 15 (but it may start earlier).

c) A translation termination signal (i.e. STOP codon) must also be included in the coding sequence for the fusion protein.

 

THE OUTLINE OF YOUR ANSWER SHOULD BE AS FOLLOWS:

Part A -Outline of sequence of steps necessary to create clone i) Indicate what restriction enzyme(s) you would use to cut the vector, and whether you would add CIP or not. Explain why

ii) Indicate what restriction enzyme(s) you would use to cut the protein Y cDNA, and whether you would add CIP or not. Explain why.

iii) Indicate what enzyme you would add to ligate the cut vector and protein Y insert together.

 

Part B Write out the sequence of the junction from i) the vector to the insert on the left end and ii) from the insert to the vector on the right end to show that your cloning strategy meets the criteria listed above. Use different colours to mark DNA that is from the vector vs. DNA that is from the insert (cDNA).

 

Part C State whether the cloning is directional or not and explain why. (Directional cloning means the insert will only go into the cut vector in one orientation. If the cloning is not directional , then that means the insert can go into the cut vector in two possible orientations.)

 

 
 

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