Q.Diagrammatically represent the experimental steps in cloning and expressing an human gene (say the gene for growth hormone) into a bacterium like E. coli?

The NCERT describes the production of human insulin in E. coli as the classic example of expressing a human gene in bacteria, and the same principle applies to cloning any human gene like growth hormone. The process begins with isolating the gene of interest—either by cutting it from human DNA using restriction enzymes or synthesizing it as cDNA from mRNA. This gene is then inserted into a plasmid vector (the cloning vehicle) that has been cut open with the same restriction enzyme, creating complementary sticky ends. DNA ligase seals the gene into the plasmid, forming recombinant DNA.

The recombinant plasmid is introduced into E. coli cells through transformation, typically using heat shock or electroporation. Only a few bacteria take up the plasmid, so selection is necessary—the plasmid carries an antibiotic resistance gene, and growing the bacteria on antibiotic-containing medium ensures only transformed cells survive. These transgenic bacteria are then cultured in large fermenters where they multiply and express the human gene, producing the desired protein (growth hormone in this case). Finally, the protein is extracted from the bacterial culture, purified, and processed for medical use.

Diagrammatic representation:

1. Isolation → Human DNA → Cut with restriction enzyme → Gene for growth hormone isolated …

The production of human proteins in bacteria is one of biotechnology's landmark achievements. Before recombinant DNA technology, proteins like insulin and growth hormone had to be extracted from human cadavers or animal sources—expensive, scarce, and sometimes unsafe. The ability to "teach" a bacterium to manufacture a human protein changed medicine entirely.

The process rests on a simple but powerful idea: if you can insert a functional human gene into a bacterial cell, and if that gene comes with the right control switches, the bacterium will transcribe and translate it just as it does its own genes. The result is a microbial factory churning out human protein.

The experimental workflow

Step 1: Isolation of the human gene

The first task is to obtain the gene for human growth hormone (hGH). Two main routes exist:

• From mRNA: Extract mRNA from human pituitary cells (which naturally produce growth hormone), then use the enzyme reverse transcriptase to synthesize complementary DNA (cDNA). This cDNA is a clean, intron-free copy of the gene—critical because bacteria lack the splicing machinery to remove introns.
• Chemical synthesis: If the gene sequence is known, it can be synthesized chemically in the lab.

The isolated gene is then amplified using PCR (polymerase chain reaction) to generate millions of copies.

Step 2: Preparation of the vector

A plasmid—a small, circular DNA molecule that replicates independently inside bacteria—serves as the vector. The plasmid is cut open at a specific site using a restriction endonuclease (a molecular scissor that recognizes a particular DNA sequence). The same enzyme is used to cut the hGH gene, ensuring that both have complementary "sticky ends."

The plasmid must carry:

• An origin of replication (ori), so it can multiply inside the host.
• A selectable marker (often an antibiotic resistance gene, like ampicillin resistance), to identify bacteria that have taken up the plasmid.
• A cloning site where the foreign gene will be inserted.

Step 3: Ligation

The hGH gene and the cut plasmid are mixed with DNA ligase, an enzyme that seals the sugar-phosphate backbone. The result is a recombinant plasmid carrying the human gene.

Step 4: Transformation

The recombinant plasmids are introduced into E. coli cells. Bacteria do not naturally take up foreign DNA, so they must be made "competent"—usually by treating them with calcium chloride and applying a brief heat shock, which makes the membrane temporarily permeable.

Only a fraction of cells will actually take up a plasmid, and among those, only some will carry the recombinant plasmid (others may carry re-ligated vector without the insert).

Step 5: Selection of transformants

The bacterial culture is plated on a medium containing an antibiotic (say, ampicillin). Only cells that have taken up a plasmid—recombinant or not—will survive, because the plasmid carries ampicillin resistance.

To distinguish recombinant clones from non-recombinant ones, a second screening step is used. If the cloning site was within the lacZ gene, recombinant colonies (where lacZ is disrupted) will appear white on X-gal medium, while non-recombinant colonies (with intact lacZ) turn blue. This blue-white screening quickly identifies the desired clones.

Step 6: Culturing and expression

Selected recombinant bacteria are grown in large fermenters under controlled conditions. As they multiply, they transcribe the hGH gene and translate it into protein. The bacteria are then harvested, lysed (broken open), and the human growth hormone is purified from the cellular extract. …

Method — a numbered flow, not prose

1. Isolate the human gene (restriction enzymes, or synthesise as cDNA from mRNA).
2. Cut a plasmid vector with the same restriction enzyme → matching sticky ends.
3. Join gene + vector with DNA ligase → recombinant plasmid.
4. Transform E. coli with the recombinant plasmid (heat shock/electroporation). …