Question

Why are plasmids and bacteriophages used as cloning vectors ?

Answer 1

Concept: Cloning vectors are DNA molecules used as vehicles to carry foreign genetic material into another cell, where it can be replicated or expressed. Plasmids and bacteriophages make excellent vectors because they can replicate on their own inside host cells.

Step 1: Explaining the functional properties of plasmids and bacteriophages.
Plasmids are small, circular, double-stranded extra-chromosomal DNA molecules found naturally in bacteria. Bacteriophages are viruses that infect bacteria and naturally inject their genetic material into the host cell.
• Both vectors possess an autonomous replication mechanism, meaning they can copy themselves independently of the host's main genomic DNA.
• Bacteriophages naturally maintain high copy numbers within bacterial cells due to their aggressive replication cycles.
• Plasmids can also replicate into multiple copies (ranging from 15 to 100 or more copies per cell). By splicing a target foreign DNA fragment into these vectors, scientists can replicate the inserted DNA at the same rate as the vector, producing many copies of the gene.

Answer 2

Concept: The synthetic plasmid vector pBR322 contains specific genetic elements, including an origin of replication, selectable antibiotic resistance markers, and unique restriction sites where restriction enzymes can cut the DNA.

Step 1: Identifying the genetic element controlling copy number.
The region labeled {ori stands for the Origin of Replication. This specific DNA sequence is where replication begins. Any foreign DNA linked to this sequence will be replicated along with the vector. Importantly, the ori site contains the control mechanisms that determine how many copies of the plasmid are made inside the host cell.

Step 2: Identifying the restriction site located within the 'rop' gene.
The rop gene codes for the proteins involved in regulating plasmid replication. Looking at the structural map of the cloning vector pBR322, the unique restriction endonuclease recognition site located within the rop gene sequence is PvuII.

Answer 3

Concept: A selectable marker is a gene introduced into a vector that allows scientists to distinguish between transformed host cells (those that have taken up the vector) and non-transformed cells, usually by providing resistance to specific antibiotics.

Step 1: Identifying the selectable marker genes in pBR322.
In the pBR322 plasmid vector, two genes provide resistance to specific antibiotics and serve as selectable markers:
• $amp^R$: This gene provides resistance to the antibiotic ampicillin.
• $tet^R$: This gene provides resistance to the antibiotic tetracycline. These genes allow researchers to select for transformed cells. If a foreign gene is inserted into one of these antibiotic resistance sites, it disrupts the gene—a process called insertional inactivation. This allows scientists to differentiate between recombinant plasmids (which contain the foreign gene) and non-recombinant plasmids.

Answer 4

Concept: Restriction enzymes recognize and cut specific DNA sequences called restriction sites. In cloning vectors, these sites are placed within selectable marker genes so that inserting foreign DNA disrupts the gene, making it easy to identify recombinant plasmids.

Step 1: Locating restriction sites within the ampicillin resistance gene ($amp^R$).
Looking closely at the genetic map of the pBR322 plasmid, the region containing the ampicillin resistance gene ($amp^R$) has recognition sites for two specific restriction endonucleases:
• PstI
• PvuI

Step 2: Locating restriction sites within the tetracycline resistance gene ($tet^R$).
Similarly, the region containing the tetracycline resistance gene ($tet^R$) has unique recognition sites for another pair of restriction endonucleases:
• BamHI
• SalI