What are plasmids? Where are they found? Why are they important to the practice

Question

What are plasmids? Where are they found? Why are they important to the practice of genetic engineering? 1. 2. Do plasmids have an importance beyond the practice of eenetic engineering? Explain. 3. What are restriction enzymes? What is the "origin of replication" on the plasmid and why is it important to the genetic engineering process? 4. What is the purpose of the antibiotic resistance genes (genes that codes for resistance to specific antibiotics) in the plasmid? Hint: The antibiotic resistance genes are used for screening purposes. Explain how they would aid in this process. 5. Why would you want your restriction enzyme to cut as close as possible to the insulin gene without cutting into it? 6.

Explanation / Answer

Ans. #1. Plasmids: The autonomously replicating extra-chromosomal circular DNA present in a bacterial cell is called plasmid. They are generally found in many eubacteria species and in few yeast species.

Plasmids are important to the practice of genetic engineering because they are used as vectors (carrier of) desired gene of interest.

#2. Yes, in interest of human beings and the environment, the degradative plasmid confers the host bacterium the ability to metabolize unusual molecules (like toluene, salicylic acid, etc.). Example- TOL plasmid of Pseudomonas putida.

#3. The endonucleases (DNA cutting enzyme) that cut/restrict DNA at specified DNA sequence are called restriction endonucleases.

#4. The “origin of replication” on the plasmid is the specific DNA segment where replication starts. For genetic engineering purpose, the vectors/plasmids must have their own origin of replication in order to replicate autonomously in the host cell.

#5. The purpose of antibiotic resistance gene in the plasmid is “Selection of recombinant cells”.

For example, a plasmid with ampR (ampicillin resistance) gene in it provides ampicillin resistance to the host cell. When a gene of interest is ligated into ampR region, the host cell loses its resistance against the antibiotic. When inoculated on a culture media with ampicillin (using replica plate technique), the transformed cells do NOT grow on this media, therefore marking the location of transformed cells on original plate.

#6. We want to isolate the whole intact insulin gene to ligate it onto a suitable plasmid and produce insulin at industrial scale. So, the restriction enzyme must cut the DNA closest to the insulin gene to avoid expression of unwanted DNA segments. Moreover, the restriction must be close to the insulin gene but not in between the gene because it would destroy the gene which would be of no use in RDT practices.

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