Question

you are asked to make a new recombinant DNA construct that expresses alkaline phosphatase protein. She just gave you a small quantity of genomic DNA and a pCDNA 3(+) vector. Using the techniques that you learned in the lab, how would you create a new recombinant plasmid?

Answer 1

pCDNA 3+ is a mammalian expression vector. To prepare a recombinant plasmid that would express the alkaline phosphatase protein :

1. The genomic DNA must be inserted into the expression vector in order to be propagated to the plasmid.
2. The genomic DNA is treated with the restriction enzymes that would cleave off the region that are not needed
3. This would prepare a DNA fragment that will contain the gene of interest which is the gene for the alkaline phosphatase.
4. The same restriction enzymes will be used to digest a portion of the expression vector to form sticky ends
5. The gene of interest containing DNA fragment is then ligated into the expression vector
6. The expression vector is then introduced into a plasmid that can be expressed in a cell
7. The pCDNA 3+ has ampicillin resistant genes and now has the gene for alkaline phosphatase enzyme
8. Hence the transformed plasmid is the introduced in a cell where the mechanism for the expression of the enzyme is present.
9. The cells are then plated on medium containing ampicillin as well as containing p-Nitrophenyl Phosphate which is the most prominent substrate for the enzyme ALP.
10. If the cells are able to transcribe the gene for the enzymes present in the plasmid, the substrate would be degraded in presence of the enzyme.
11. There will yellow coloration in the media which will prove that the substrate has been reacted upon by the enzyme