Biotechnology Workshop Week 6

Restriction Enzymes

• Proteins that cut DNA at specific sequences.
• Make two cuts in double-stranded DNA (dsDNA) creating blunt ends (straight cuts) or sticky ends (staggered cuts with overhangs).
• Useful in genetic engineering and molecular biology for fragment generation.

Cloning the Human Insulin Gene

• Isolate the insulin gene by extracting mRNA from human pancreatic cells and converting it to complementary DNA (cDNA).
• Prepare a vector by cutting a bacterial plasmid with a restriction enzyme to create sticky ends.
• Ligate the insulin cDNA into the plasmid, creating a recombinant plasmid.
• Transform bacterial cells with the recombinant plasmid and select for successful transformants.
• Screen bacterial clones to confirm the presence of the insulin gene.
• Induce growth and purify the produced insulin protein.

Characteristics of a Cloning Vector

• Origin of Replication (ori): Enables independent replication within the host cell.
• Selectable Marker: Typically an antibiotic resistance gene, allowing identification of cells that have taken up the vector.
• Multiple Cloning Site (MCS): Contains unique restriction enzyme sites for easy insertion of foreign DNA.

DNA Gel Electrophoresis

• Separation Characteristics: DNA fragments are separated based on size and charge.
• Migration Process: DNA samples loaded in a gel move towards the positive electrode; smaller fragments migrate faster and further, forming distinct bands.
• Visualization: Ethidium bromide (EtBr) is commonly used, intercalating with DNA and fluorescing under UV light for band visibility.

Selectable Markers in Plasmids

• Antibiotic Resistance Gene: Provides survival advantage on antibiotic media, enabling selection of transformed bacteria.
• LacZ Gene: Codes for β-galactosidase, turning blue in the presence of X-gal; white colonies indicate successful gene insertion.

Types of Vectors

• Bacterial Artificial Chromosomes (BACs): Can carry large fragments (up to 300,000 base pairs); ideal for cloning extensive DNA segments in bacterial cells.
• Yeast Artificial Chromosomes (YACs): Larger capacity (up to 1 million base pairs); useful for cloning very large DNA sequences in yeast cells.

Differences Between Expression and Cloning Vectors

• Purpose: Cloning vectors amplify DNA; expression vectors produce proteins encoded by genes.
• Regulatory Elements: Cloning vectors have basic elements; expression vectors include promoters and binding sites for protein synthesis.
• End Product: Cloning vectors yield multiple DNA copies; expression vectors yield the protein product.

Drawbacks of Bacterial Expression Vectors for Human Genes

• Lack of Post-Translational Modifications: Bacteria cannot perform complex modifications (e.g., glycosylation), essential for human protein function.
• Protein Misfolding and Aggregation: Human proteins may misfold or aggregate in bacteria, resulting in non-functional proteins.