Recombinant DNA Technology & Genetic Engineering

• The module discusses recombinant DNA technology and genetic engineering and their applications.

Objectives

• Outline the steps in molecular cloning
• Describe methods to introduce DNA into cells
• Explain the selection and screening of genetically modified organisms (GMOs)
• Discuss some applications of recombinant DNA

Subject Content

• Genetic Engineering: Development and application of scientific methods to manipulate genetic material to alter hereditary traits.
• Biotechnology: Using biological processes/organisms to manufacture products that improve human life.
• Recombinant DNA: DNA with inserted segments or genes from different molecules, creating a new genetic combination.

Classical vs. Modern Genetic Techniques

• Classical breeding involves mating organisms w/ desirable qualities to produce offspring with favorable traits.

Classical Breeding

• Widely used for food crops to improve food quality, yield, tolerance, resistance to environmental threats, and shelf life.
• Disadvantage: it takes time due to the randomness of meiosis.

Modern Techniques

• They involve molecular techniques to modify traits by:
  • Introducing new traits
  • Increasing gene expression,
  • Disrupting gene expression inhibition

Examples of Modified Traits and Techniques

• Kobe/Wagyu Beef: Classical breeding is used to achieve good fat distribution.
• Guapple: Classical breeding produce large-sized guavas.
• Human Insulin-producing bacteria: Recombinant DNA Technology is used to produce them.
• Flavr-Savr Tomatoes: Recombinant DNA Technology delays ripening.
• Macapuno trait in coconuts is achieved through Classical breeding.

General Outline of Recombinant DNA Technology

• Cutting the desired gene by restriction enzymes.
• Selecting an appropriate vector to propagate the gene.
• Ligation: Joining the desired gene and the vector's gene.
• Transferring the desired gene to the vector for reproduction

Further Steps

• Selecting vectors able to contain the new gene.
• Sequencing to make sure the proper product happens

Plasmid DNA in Bacteria

• Plasmids: Shorter, circular DNA sequences replicated by bacteria
• Great targets for genetic modification

Creating the Desired Plasmid

• Restriction enzymes are utilized
• Restriction enzymes are DNA "scissors" that cut DNA at specific sequences
• EcoRI cuts along GAATT

EcoRI Function

• The restriction enzyme cuts the plasmid and desired gene.
• Both DNA will have a sticky end of AATT on one direction and TTAA on the other.
• AATT and TTAA ends are complementary, allowing ligation to form the new plasmid.

Human Insulin Production

• Process showing how recombinant plasmids are introduced into bacteria to produce human insulin.

Ways of Introducing Desired Gene to Host Organisms

• Heat Shock Treatment: Target cells are pre-treated to increase membrane pore sizes using CaCl2 to make cells competent.
• Incubate cells with the plasmid at 4°C, then heat shock at 42°C to allow plasmid to enter.
• Cells taking up plasmids are "transformed."
• Biolistics: A gene gun is used to fire DNA-coated pellets on plant tissues.
• Electroporation: Electric shock is used to expand the membrane pores, for insertion of genes into mammalian cells.

Screening Recombinant Cells

• Using an ampicillin resistance gene (AMP) as a selection marker.
• Only "transformed" cells survive in ampicillin, allowing them to grow into colonies.

Selection of Transformed Cells

• Inserted genes can provide visible proof, like antibiotic resistance, colored proteins, or fluorescent products (e.g., GFP).

PCR Detection of Plasmid DNA

• PCR amplification confirms the presence of the desired gene in inserted plasmids

Genetically Modified Organisms (GMOs)

• The ability to insert gene sequences to provide new traits.
• Improving product yield

The Flavr-Savr Tomato

• 1st genetically modified organism for human consumption.
• A antisense-RNA disrupts the expression of polygalacturonase that delays softening of the fruit.

Bt-Corn

• Corn incorporates the Bt-endotoxin from Bacillus thuringiensis .
• The toxin results in the death of pests, non-toxic to humans, mammals or fish.
• Increases crop production
• Same technology is applied for Bt-Eggplant in the Philippines