Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?
A reporter enzyme like β-galactosidase helps in monitoring transformation by differentiating recombinants from non-recombinants based on colour production in the presence of a chromogenic substrate.
In this method, the coding sequence of the β-galactosidase gene contains the insertion site for the foreign DNA. When the foreign DNA is inserted within this gene, it results in insertional inactivation of the enzyme.
When grown on a medium containing a chromogenic substrate, non-recombinant bacteria (without insert) produce blue-coloured colonies as the β-galactosidase gene remains functional. However, recombinant bacteria (with insert) produce colourless colonies since the gene gets inactivated due to insertion of alien DNA. This allows easy visual identification of transformed cells.
Explanation
The textbook explains that alternative selectable markers have been developed which differentiate recombinants from non-recombinants based on colour production. The β-galactosidase gene serves as a reporter enzyme. When foreign DNA is inserted within its coding sequence, insertional inactivation occurs. In the presence of a chromogenic substrate, functional β-galactosidase produces blue colour (non-recombinants), while inactivated enzyme produces no colour (recombinants). This is more efficient than antibiotic-based selection which requires simultaneous plating on two different antibiotic plates.
Solution Steps
Step 1: Insert foreign DNA within the coding sequence of β-galactosidase gene in the vector
Step 2: Transform host cells and grow them on medium containing a chromogenic substrate
Step 3: Observe colony colour - blue colonies indicate non-recombinants, colourless colonies indicate recombinants (successful transformation)