4.4 Transformation and selection methods

Genetic engineering relies on getting DNA into cells and identifying which cells took it up. Scientists use clever tricks like zapping cells with electricity or heat to make them absorb DNA. Then they use special genes that help the transformed cells survive or stand out.

These methods are crucial for creating genetically modified organisms. By introducing foreign DNA and selecting for successful transformants, researchers can study gene function, produce valuable proteins, and develop new biotechnology applications.

Transformation Methods

Introducing Foreign DNA into Bacterial Cells

Top images from around the web for Introducing Foreign DNA into Bacterial Cells

• 

  Cloning | Biology for Non-Majors I View original

  Is this image relevant?

• 

  Frontiers | Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation View original

  Is this image relevant?

• 

  Microbes and the Tools of Genetic Engineering | Microbiology View original

  Is this image relevant?

• 

  Cloning | Biology for Non-Majors I View original

  Is this image relevant?

• 

  Frontiers | Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation View original

  Is this image relevant?

1 of 3

Top images from around the web for Introducing Foreign DNA into Bacterial Cells

• 

  Cloning | Biology for Non-Majors I View original

  Is this image relevant?

• 

  Frontiers | Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation View original

  Is this image relevant?

• 

  Microbes and the Tools of Genetic Engineering | Microbiology View original

  Is this image relevant?

• 

  Cloning | Biology for Non-Majors I View original

  Is this image relevant?

• 

  Frontiers | Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation View original

  Is this image relevant?

1 of 3

• Bacterial transformation involves introducing foreign DNA into bacterial cells to create recombinant DNA molecules
• Requires making bacterial cells competent, meaning temporarily increasing their cell membrane permeability to allow uptake of foreign DNA
• Competent cells are prepared by treating them with chemicals (calcium chloride) or by applying an electric field (electroporation)
• Once cells are competent, they can take up foreign DNA from their surrounding environment
• After transformation, cells are grown on selective media to identify and isolate those that have successfully incorporated the foreign DNA

Electroporation and Heat Shock

• Electroporation uses high-voltage electric pulses to create temporary pores in the cell membrane, allowing DNA to enter the cell
  • Cells are mixed with DNA in a special buffer and placed in a cuvette between two electrodes
  • A brief electric pulse (5-10 milliseconds) at a high voltage (1,000-2,500 volts) is applied, causing the formation of temporary pores in the cell membrane
  • DNA enters the cell through these pores, and the pores quickly reseal once the electric field is removed
• Heat shock is another method for introducing DNA into bacterial cells
  • Cells are mixed with DNA and calcium chloride, which neutralizes the negative charges on the cell surface and DNA, allowing the DNA to adhere to the cell
  • The mixture is then briefly heated to 42°C for 30-60 seconds, causing the cell membrane to become more fluid and permeable
  • The cells are then quickly cooled on ice, causing the membrane to solidify and trap the DNA inside the cell

Selection Techniques

Antibiotic Resistance and Selectable Markers

• Antibiotic resistance markers are genes that confer resistance to specific antibiotics, allowing transformed cells to grow in the presence of the antibiotic
  • Common antibiotic resistance markers include ampicillin, kanamycin, and chloramphenicol resistance genes
  • The antibiotic resistance gene is usually included in the vector along with the gene of interest
  • After transformation, cells are plated on media containing the appropriate antibiotic, and only cells that have taken up the vector with the resistance gene will survive and form colonies
• Selectable markers are genes that allow transformed cells to grow under specific conditions, such as in the presence of a particular nutrient or toxin
  • Examples include the

    lacZ

    gene, which allows cells to metabolize lactose, and the

    ura3

    gene, which allows yeast cells to grow in the absence of uracil
  • Selectable markers are often used in combination with antibiotic resistance markers to increase the efficiency of selecting transformed cells

Screening Techniques for Identifying Recombinant Clones

• Blue-white screening is a technique used to visually identify bacterial colonies that contain recombinant plasmids
  • The method relies on the

    lacZ

    gene, which encodes the enzyme β-galactosidase that cleaves the substrate X-gal, producing a blue color
  • The vector contains a multiple cloning site within the

    lacZ

    gene, and when a foreign DNA fragment is inserted, it disrupts the

    lacZ

    gene and prevents the production of functional β-galactosidase
  • Colonies containing the recombinant plasmid will appear white, while those with the intact

    lacZ

    gene will be blue
• Reporter genes are genes that produce an easily detectable product, such as a fluorescent protein or an enzyme, when expressed
  • Examples include the green fluorescent protein (GFP) and luciferase genes
  • Reporter genes are often fused to the gene of interest in the vector, allowing researchers to monitor the expression of the target gene by measuring the activity of the reporter
• Colony PCR is a method for quickly screening a large number of bacterial colonies for the presence of a specific DNA sequence
  • A small amount of bacterial cells from each colony is used directly as a template for PCR amplification
  • Primers specific to the gene of interest are used, and the presence of the correct PCR product indicates that the colony contains the desired recombinant plasmid
  • Colony PCR is a rapid and efficient way to identify positive clones without the need for plasmid isolation or restriction digestion