Discuss the following questions with your partner and be prepared to share your

Question

Discuss the following questions with your partner and be prepared to share your answers with the class.

1. List in words or indicate in a drawing the important features of a plasmid vector that are required to clone a gene. Explain the purpose of each feature.

2. What role do restriction enzymes have in nature?

3. Using your understanding of evolution, why would bacteria retain a gene that gives them resistance to antibiotics? How is the existence of bacteria with antibiotic resistance affecting medicine today?

4. Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria.

5. Due to a mishap in the lab, bacteria carrying a plasmid with a kanamycin- resistant gene and bacteria carrying a plasmid with an ampicillin-resistant gene were accidentally mixed together. Design an experiment that will allow you to sort out the two kinds of bacteria. (Hint: Make sure that you do not kill off one of the kinds of bacteria you are trying to sort out!)

Explanation / Answer

1. The plasmid vectors shall have the following features: Origin of replication, a marker gene (best suited if the gene product results in complementation than antibiotic resistance), unique restriction sites within the marker gene, high copy number of the plasmid.

2. Restriction enzymes offer protection to bacteria from bacteriophages. They modify the host DNA in such a way, the host DNA remain unaffected while the infecting phage DNA is specifically degraded.

3. Antibiotic resistance is not an evolution, it results from selection. In a population of bacterial cells, randon mutations lead to one or few of cells in millions of bacteria to have a mutated gene that may confer them with antibiotic resistance. When the population are exposed to an antibiotic, all the susceptible cells die, leaving the resistant bacteria to survive. These surviving bacteria reproduce and all their offspring are resistant to the given antibiotic. The same selection process repeats over time that give rise to bacteria with multiple drug resistance.

Bacteria with antibiotic resistance pose problems in treatment, as they are difficult to be killed. The more is the drug resistance, the more is the difficulty to treat a disease caused by that bacteria.

4. Genes from humans are different from those of bacteria. Eukaryotic genes (human genes) contain introns, which need to be removed from mRNA by splicing. Bacterial genes do not have introns, they do not have machinery for splicing introns. Also, the post transcriptional and post translational modifications occur in eukaryotes, where they are not that prominent in prokaryotes.

To express eukaryotic genes in bacteria, cDNA is to be cloned into suitable vector, not the gene. cDNA is synthesized form coding sequences of mRNA. Along with suitable modification systems, the cloned vector is inserted into the bacterial cell. Only those type of eukaryotic proteins which does not need extensive post translational modifications can be expressed in prokaryotes.

5. Replica plating method can be used to sort out the bacteria. A sample of bacteria is initialy grown on a medium with one of the antibiotics (say kanamycin). When colonies appear, replica plating technique can be applied to exaclty replicate the cells, and grow the replica in medium containing the second antibiotic.

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