Problem 11 - Campbell Biology Concepts & Connections

Question

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.)

Step-by-Step Solution

Answer

Insert the HGH gene into a plasmid, transform E. coli, select and screen transformed colonies, and then extract and purify the HGH.

Step 1 - Cut the Plasmid with Restriction Enzyme

Use a restriction enzyme to cut the plasmid DNA at a specific recognition site. This creates sticky ends on the plasmid, which will help in ligating the human HGH gene.

Step 2 - Cut the HGH Gene with the Same Restriction Enzyme

Extract the DNA carrying the human HGH gene and cut the DNA using the same restriction enzyme. This ensures that the ends of the HGH DNA are complementary to the ends of the plasmid DNA.

Step 3 - Ligate the HGH Gene into the Plasmid

Use DNA ligase to join the HGH gene with the plasmid DNA. The complementary sticky ends will anneal, and the DNA ligase will create covalent bonds to form a continuous circular plasmid.

Step 4 - Transform E. coli with Recombinant Plasmid

Introduce the recombinant plasmid into E. coli cells using methods such as heat shock or electroporation, allowing the bacteria to take up the plasmid.

Step 5 - Select and Grow Transformed E. coli

Plate the bacteria on a selective medium that contains an antibiotic. Only the E. coli that have taken up the plasmid (which should have an antibiotic resistance gene) will survive and grow.

Step 6 - Screen for Presence of HGH Gene

Use an appropriate DNA probe that is complementary to the HGH gene sequence to screen the colonies. This confirms which colonies have the HGH gene.

Step 7 - Extract and Purify HGH

Grow the selected E. coli colonies in bulk culture and then extract the human growth hormone. Purify the hormone using protein purification techniques such as affinity chromatography.

Key Concepts

recombinant DNA technology

Recombinant DNA technology is a powerful genetic engineering technique used to create DNA molecules with new genetic combinations. This method involves joining together DNA from different organisms to produce new genetic sequences. It is widely used to generate organisms capable of making proteins like human growth hormone (HGH).

For example, scientists can combine a plasmid (a small, circular piece of DNA from a bacterium like E. coli) with a gene from another organism. This forms what is called recombinant DNA, which can then be inserted into a bacterium to produce the desired protein.

restriction enzymes

Restriction enzymes, also called molecular scissors, are proteins used to cut DNA at specific sequences called restriction sites. By cutting both the plasmid and the target DNA (like the HGH gene) with the same enzyme, 'sticky ends' are created. These sticky ends are crucial for guiding the insertion of the target DNA into the plasmid.

For instance, enzymes like EcoRI recognize and cut specific sequences, creating complementary overhangs. This allows the HGH gene to precisely fit into the plasmid, ensuring efficient ligation, or joining.

DNA ligase

DNA ligase is an important enzyme that acts as a glue in genetic engineering. After the restriction enzyme creates sticky ends on both the plasmid and the HGH gene, DNA ligase joins these pieces together. The enzyme forms covalent bonds between adjacent nucleotides, stabilizing the circular plasmid.

This step is essential to ensure the human HGH gene is securely incorporated into the plasmid, which will then be introduced into E. coli bacteria for further processes.

transformation

Transformation is the process of introducing recombinant DNA into bacteria. E. coli cells are made competent to take up foreign DNA through methods like heat shock or electroporation. These techniques temporarily create pores in the cell membranes, allowing the recombinant plasmid to enter.

Once inside the E. coli, the plasmid replicates along with the bacterial DNA, resulting in a colony of bacteria each capable of producing the human growth hormone.

selective medium

A selective medium is used to grow bacteria that contain the recombinant plasmid. Typically, the plasmid is engineered to include an antibiotic resistance gene. When the transformed E. coli are grown on a medium containing that antibiotic, only bacteria that have successfully taken up the plasmid will survive.

This step ensures that only the bacteria carrying the recombinant plasmid (and thus the HGH gene) will grow, simplifying the identification and isolation of successful transformants.

DNA probe

A DNA probe is a single-stranded DNA sequence that is complementary to the target gene you want to identify. In this case, a DNA probe complementary to the HGH gene is used to screen transformed E. coli colonies. The probe binds to the HGH gene in a colony, confirming its presence.

Screening with a DNA probe ensures that the selected colonies contain the gene of interest, making subsequent steps like hormone extraction more efficient.

protein purification

Protein purification is the final stage where the human growth hormone is extracted from the cultured E. coli and purified. Techniques like affinity chromatography are often used. In this method, a column containing a material that specifically binds to HGH is used to isolate the protein from other bacterial proteins.

This purified hormone can then be used for medical treatments and other applications, completing the process of recombinant DNA technology.