You intend to use plasmid pVEC-4kb (4.4kb in size and draw n approximately to sc

Question

You intend to use plasmid pVEC-4kb (4.4kb in size and draw n approximately to scale below) as a vector for inserting LDH ORF sequence, to be cloned in E. coli. Below is the map of the plasmid. which shows the locations of restriction sites of various restriction endonucleases. the origin of replication (ORI) and Ampicilin-resistance gene (Amp^t). a) Describe what Amp' and ORI arc and their importance' to the plasmid. b) List the pair(s) of restriction endonucleases you would use to digest your plasmid to be able to insert the LDH coding sequence and to clone it in E. coli. Explain why you picked your specific pair(s) of endonucleases. Briefly explain why you did not choose other pairs. c) What must you have in the insert sequence to accomplish this goal? Be specific and tell exactly how you would accomplish this Depict your understanding by drawing a sketch. d) How would you control directionality of insertion of the LDH sequence? Be specific and tell exactly what you would do and show your understanding with a sketch.

Explanation / Answer

a.

Ampr indicates ampicillin resistnace. The gene confers ampicillin resistance and hence, organisms that possess the plasmid can grow in the presence of ampicillin.

ORI is DNA region that signals for origin of replication. This is the place where the plasmid replication begins. The regions of ORI are generally rich in Adenine and Thymine, since A-T rich regions melt more readily and facilitates the replication machinery.

b.

To clone the given gene, the plasmid is digested with PstI and HaeIII enzymes, and the same is the case with the gene of interest also. Digestion with the said genes has two advantages: The digested plasmid do not recircularize since the two sticky ends generated have different base compositions and are not complementary to each other. Therefore, autorecircularization of the plasmid is avoided.

The second advantage is that Ampr gene is a selectable marker. If cloning of foreign DNA is done at this site, resistance or sensitivity to ampicillin serves as a marker for identification of clones that have the foreign gene insert. If the plasmid does not has the foreign gene inserted, it is resistant to the antibiotic added in the growth medium. On the other hand, if there is a foreign insert, the ampicillin resistance gene is removed and hence, the organisms become susceptible to the antibiotic in the growth medium.

This enables easy selection of host cell colonies that have the foreign gene inserted.

The other sequences are not chosen for the following reasons:

c.

The gene of interest also must be digested by the same combination of restriction enzymes in order to insert the gene into the plasmid.

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