Explain how you might engineer E. coli to produce human growth hormone (HGH) using the following: E. coli containing a plasmid, DNA carrying the gene for HGH, DNA ligase, a restriction enzyme, equipment for manipulating and growing bacteria, a method for extracting and purifying the hormone, and an appropriate DNA probe. (Assume that the human HGH gene lacks introns.)

Verified step by step guidance

1. Step 1: Use a restriction enzyme to cut the plasmid DNA from E. coli and the DNA carrying the human growth hormone (HGH) gene. Restriction enzymes recognize specific sequences in DNA and create cuts, often leaving 'sticky ends' that can facilitate the joining of DNA fragments.
2. Step 2: Combine the cut plasmid DNA and the HGH gene DNA. The sticky ends created by the restriction enzyme will allow the two DNA fragments to align and bond. Use DNA ligase to seal the sugar-phosphate backbone, creating a recombinant plasmid that contains the HGH gene.
3. Step 3: Introduce the recombinant plasmid into E. coli cells through a process called transformation. This can be achieved by making the bacterial cells competent (e.g., using heat shock or electroporation) to take up the plasmid DNA.
4. Step 4: Grow the transformed E. coli cells in a nutrient-rich medium. The bacteria will replicate, and as they do, they will also replicate the recombinant plasmid, leading to the production of human growth hormone. Ensure the plasmid includes a promoter sequence that allows the HGH gene to be expressed in the bacterial cells.
5. Step 5: Extract and purify the human growth hormone from the bacterial culture. Use a method such as affinity chromatography to isolate the hormone. To confirm the presence of the HGH gene in the plasmid, use an appropriate DNA probe that hybridizes specifically to the HGH gene sequence.

Key Concepts

Recombinant DNA Technology

Recombinant DNA technology involves combining DNA from different sources to create new genetic combinations. In this context, it allows for the insertion of the human growth hormone (HGH) gene into a plasmid, which can then be introduced into E. coli. This process enables the bacteria to express the HGH protein, which can be harvested for therapeutic use.

Plasmids and Cloning Vectors

Plasmids are small, circular DNA molecules that can replicate independently within a bacterial cell. They are commonly used as cloning vectors to carry foreign genes, such as the HGH gene, into host organisms like E. coli. The plasmid must contain a promoter for gene expression and a selectable marker to identify successfully transformed cells.

Gene Expression and Protein Purification

Gene expression refers to the process by which the information in a gene is used to synthesize a functional protein. After E. coli has been transformed with the plasmid containing the HGH gene, the bacteria will produce HGH. The final step involves extracting and purifying the hormone from the bacterial culture, which may include techniques like affinity chromatography to isolate the HGH protein from other cellular components.