Question

What is the correct sequence of steps in the genetic engineering of {E.coli for insulin production?
A. Obtaining a copy of the human insulin gene by isolating mRNA
B. Switching on gene action.
C. Identifying transformed bacteria prior to cloning
D. Inserting the DNA into a plasmid vector and inserting the plasmid vector into the host bacterium}
Choose the most appropriate answer from the options given below:

• A. A,B,C, D
• B. A,C,B,D
• C. A,D,C,B
• D. C,B,D,A

Hint:

Think of the process like a recipe:

\textbf{Get the gene} (Isolate the recipe - Step A).
\textbf{Put the gene in a carrier and get it into the factory} (Put the recipe in a cookbook and give it to the chef - Step D).
\textbf{Find the factory that accepted the gene} (Make sure the chef got the right cookbook - Step C).
\textbf{Tell the factory to start producing} (Tell the chef to start cooking - Step B).

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
The question asks for the correct workflow for producing a human protein (insulin) in a bacterial host ({E. coli}) using recombinant DNA technology.

Step 2: Detailed Explanation of the Sequence:
1. A. Obtaining a copy of the human insulin gene by isolating mRNA: This is the first essential step. The gene for insulin needs to be isolated from a human source. In practice, mRNA from pancreatic cells is isolated and used as a template for the enzyme reverse transcriptase to create a complementary DNA (cDNA) copy of the gene. This cDNA is used because it lacks introns, which bacteria cannot process.
2. D. Inserting the DNA into a plasmid vector and inserting the plasmid vector into the host bacterium: Once the insulin gene (cDNA) is obtained, it is inserted (ligated) into a plasmid vector. This recombinant plasmid is then introduced into the host bacteria ({E. coli}) through a process called transformation.
3. C. Identifying transformed bacteria prior to cloning: Transformation is not 100% efficient. Therefore, it is crucial to select or identify the bacteria that have successfully taken up the recombinant plasmid. This is often done using selectable markers, such as antibiotic resistance genes, that are also on the plasmid. Only the transformed bacteria will grow on a medium containing the antibiotic.
4. B. Switching on gene action: After selecting the transformed bacteria, they are cultured in large quantities (cloned). To produce the insulin protein, the expression of the inserted gene must be induced or "switched on." This is often controlled by an inducible promoter in the plasmid, which can be activated by adding a specific chemical (like IPTG) to the culture medium.

Step 3: Final Answer:
The logical and correct sequence of steps for producing recombinant insulin is A \(\rightarrow\) D \(\rightarrow\) C \(\rightarrow\) B.