Question

Question 1 - 12 What does the AMPr gene in the PGEM-T plasmid produce and what is the role of this product in the protocol itself? Type your answer Question 2 --12 What does the lacZ gene in the PGEM- T plasmid produce and what is the role of this product in the protocol itself? Type your answer Question 3 (413) What occurs during the ligation step of Protocol 11? Type your answer Question 4 ( -- /2 What is the underlying reason for transforming bacteria with this modified PGEM-T plasmid?

Answer 1

1. Ampicillin resistance gene = TEM-1 β-lactamase gene
It produces an enzyme that breakdown Ampicillin.
The role of the TEM-1 β-lactamase gene in this protocol is that only transformed cells can grow in the presence of the antibiotic.
This allows us to eliminate untransformed cells.

2. LacZ gene codes for β-galactosidase. It converts colorless IPTG+X-gal solution to a blue-colored product.
The MCS is located within the LacZ coding sequence.
So, self-ligated vectors reconstitute the lacZ coding sequence. Such false-positive colonies appear in blue color.
Recombinant vectors inactivate the lacZ coding sequence due to the integration of the insert. Such true recombinant colonies appear in white color.

3. During the ligation step, a phosphodiester bond is formed between two compatible DNA ends.
This process is mediated by ATP-dependent DNA ligase.

4. To produce recombinant proteins.