Recombination and Transformation

Restriction Enzymes

Restriction Enzymes

Enzymes that cut DNA at specific recognition sites, creating blunt ends or sticky ends.

Palindromic Restriction Site

A region of DNA with bases that read the same in a 5’ to 3’ direction on both strands.

Endonuclease

Enzyme that cleaves a polynucleotide chain, cutting upstream and downstream of a gene to leave sticky ends.

Plasmid

Small loops of DNA from bacteria used in genetic engineering to carry genes.

Recombinant Plasmid

Result of incorporating a foreign gene into a plasmid using restriction enzymes and ligase.

Transformation

Process of making bacteria competent to take up plasmids, identifying transformed bacteria on antibiotic plates.

Human Insulin

Protein with A and B chains held by disulfide bonds, produced by combining cloned polypeptides in vitro.

Genetic Code Redundancy

Multiple codons coding for the same amino acid, allowing for different nucleotide sequences to code for the same protein.

LacZ Gene

Gene encoding Beta-galactosidase, used in genetic engineering to identify transformed bacteria by turning blue in the presence of X-gal.

LacZ gene

Used in recombinant plasmids to produce β-Galactosidase, aiding in the identification of colonies with the recombinant plasmid due to the blue color produced.

Fusion gene

Two genes sharing a single promoter, transcribed and translated together without a stop codon between them.

Methionine addition

Added to the start of the insulin A gene to facilitate the separation of the fusion protein once out of the cell.

Recombinant plasmids

Formed by mixing DNA fragments from human insulin with a plasmid containing antibiotic-resistance genes, joined by DNA ligase.

Transformation

Process of introducing plasmids into E. coli bacteria, where only some bacteria pick up the plasmids, leading to transformed bacteria.

Selection of bacteria

Transformed bacteria are cultured on agar plates containing antibiotics like ampicillin and tetracycline to select for those carrying the recombinant plasmid.

Manufacture of human insulin

Transformed bacteria with the recombinant plasmid are cultured in fermenters to replicate the plasmid and produce polypeptides, which are then processed into functional human insulin.