Objective Select from different restriction enzyme sites to

Objective: Select from different restriction enzyme sites to determine which are best for a particular cloning procedure 1.You are interested in studying some features of the NF-KB gene, which is involved in cellular responses to stimuli such as stress. To study the gene, you first need to be able to generate it in large quantities, and then potentially manipulate it. So, you begin by cloning the gene into a plasmid (the circular piece of bacterial DNA shown below on the left). AmpR represents the ampicillin resistance gene. Each of the letters in the diagram represent a different restriction enzyme site. Ori B S B AmpR NFK2 gene A What is the ORI essential for? B. What will happen to the transformed bacteria if the AmpR site is disrupted (for example, by cutting with a restriction enzyme)? C. What would happen if you grew transformed bacteria transformed with the plasmid above on plates containing the antibiotic Kanamycin?

Solution

1 A. ORI i.e. origin of replication is a unique DNA sequence which is 50-100 base pairs long and is the point at which the replication begins. Hence, in a cloning vector ORI is essential for replication.

B. AmpR site i.e ampicillin resistant gene on a plasmid indicates that the bacteria containing this plasmid is resistant to ampicillin. Ampicillin resistant gene encodes a protein called -lactamase. This protein inactivates the antibiotic ampicilllin. In other words, the bacteria containing this plasmid will be able to grow in medium even in the presence of ampicillin (which is an antibiotic).Hence, after transformation, only bacteria contain a plasmid with this AmpR gene will be able grow in a medium containing ampicillin. Therefore if the AmpR site is disrupted in a plasmid, this ability to select only cells that have been transformed, will be lost.

C. On plates containing kanamycin, we will see no colonies of bacteria. This is because the transformed bacteria will contain only the ampicilllin resistant gene and not kanamycin resistant gene.

D. The enzymes S and E should be used for cutting this plasmid and gene. Enzyme B cannot be used as one of its restriction site is present in the AmpR gene. This enzyme would hence disrupt the AmpR gene and thus making transformation inefficient. The H enzyme cannot be used because one of its restriction site is present in the NFK2 gene. The gene would get disrupted in presence of this enzyme. E and S do not have any restriction sites on the AmpR gene or the NFK2 gene. For the gene to be ligated into the plasmid, complementary sticky ends at both ends are required. Hence since E and S enzymes have restriction sites flanking the NFK2 gene they can be used to cut the plasmid too , resulting in complementary sticky ends in the plasmid and gene.