Outline the process of genetic modification using bacterial production of a human protein as an example, limited to: (a) isolation of the DNA making up a human gene using restriction enzymes, forming sticky ends (b) cutting of bacterial plasmid DNA with the same restriction enzymes, forming complementary sticky ends (c) insertion of human DNA into bacterial plasmid DNA using DNA ligase to form a recombinant plasmid (d) insertion of recombinant plasmids into bacteria (specific details are not required) (e) multiplication of bacteria containing recombinant plasmids (f) expression in bacteria of the human gene to make the human protein

Sourav PanNovember 15, 2024

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Process of Genetic Modification Using Bacterial Production of a Human Protein

The following outlines the key steps involved in genetically modifying bacteria to produce a human protein, such as insulin, focusing on specific processes including the isolation and insertion of human DNA.

(a) Isolation of the DNA Making Up a Human Gene Using Restriction Enzymes

1. Gene Identification: The first step involves identifying the specific human gene that codes for the desired protein (e.g., the insulin gene).
2. DNA Extraction: The DNA is extracted from human cells (often from a tissue sample).
3. Restriction Enzyme Digestion: Restriction enzymes are used to cut the DNA at specific sequences, isolating the desired gene. These enzymes create “sticky ends,” which are short, single-stranded overhangs of DNA that facilitate the joining of DNA fragments later in the process.

(b) Cutting of Bacterial Plasmid DNA with the Same Restriction Enzymes

1. Plasmid Preparation: A plasmid (a small circular piece of DNA found in bacteria) is selected as a vector for gene insertion.
2. Restriction Enzyme Digestion: The same restriction enzymes used to isolate the human gene are employed to cut the plasmid DNA. This results in complementary sticky ends on both the plasmid and the human gene, enabling them to bond together.

(c) Insertion of Human DNA into Bacterial Plasmid DNA Using DNA Ligase to Form a Recombinant Plasmid

1. Ligation Reaction: The isolated human gene with sticky ends is mixed with the cut plasmid DNA, allowing them to anneal due to complementary base pairing.
2. DNA Ligase Action: The enzyme DNA ligase is added to catalyze the formation of covalent bonds between the sugar-phosphate backbones of the human gene and plasmid DNA, resulting in a recombinant plasmid that contains both bacterial and human DNA.

(d) Insertion of Recombinant Plasmids into Bacteria

1. Transformation Process: The recombinant plasmids are introduced into competent bacterial cells (often E. coli) through a process known as transformation. This can be achieved using heat shock or electroporation methods that facilitate the uptake of plasmids by bacteria.

(e) Multiplication of Bacteria Containing Recombinant Plasmids

1. Bacterial Growth: The transformed bacteria are placed in a nutrient-rich culture medium and incubated under optimal conditions (temperature, pH, etc.).
2. Clonal Expansion: As the bacteria multiply through binary fission, each daughter cell inherits the recombinant plasmid, leading to an exponential increase in the number of bacteria containing the human gene.

(f) Expression in Bacteria of the Human Gene to Make the Human Protein

1. Induction of Protein Expression: Once sufficient bacterial growth is achieved, conditions are adjusted (such as adding an inducer) to trigger the expression of the human gene.
2. Protein Production: The bacteria utilize their cellular machinery to transcribe and translate the inserted human gene into the corresponding protein (e.g., insulin).
3. Harvesting: After sufficient protein production, bacterial cells can be lysed to release the human protein, which can then be purified for medical use.