Central Dogma: DNA (Transcription)→ RNA (Translation)→ Protein

GFP: Green Fluorescent Protein

• found in Pacific Jellyfish(Aequorea victoria)

• When excited by UV light/radiation the protein gives off a green light

• applications:

  • genetic engineering includes the treatment of human genetic disease

  • production of transgenic crops

Transformation: Uptake of foreign DNA(often circular plasmid)

Transformation Principle:

1. Introduce foreign gene(aka recombinant molecule) into host cells(bacteria, yeast, Chinese hamster ovary)

2. Plasmid replicate inside the host, causing genes on plasmid to be expressed including selectable marker

3. Inefficient process as only 1/10,000 cells transform

Process of Bacterial Transformation:

1. Preparation of competent cells:

   1. Bacteria are treated with specific chemicals or subjected to temperature changes to make them "competent," or capable of taking up DNA.

2. Addition of foreign DNA:

   1. The desired DNA, often in the form of a plasmid, is added to the competent cells.

3. Incubation:

   1. The cells are incubated on ice to allow the DNA to bind to the cell surface.

4. Heat shock:

   1. The cells are briefly exposed to a sudden increase in temperature, which causes the cell membranes to become more permeable, allowing the DNA to enter the cells.

5. Recovery:

   1. The cells are then incubated in a nutrient-rich medium to allow them to recover and express the genes carried by the foreign DNA.

Plasmid:

• circular piece of autonomously replicating DNA

• originally evolved by bacteria

• May express antibiotic resistance genes or be modified to express proteins of interest

Gene Expression

• bla: Beta Lactamase

  • Ampicillin resistance

• GFP: Glowing Fluorescent protein (Aequorea victoria jellyfish gene)

• araC: regulator protein

  • Regulates GFP transcription

Electroporation:

• Electrical shock makes cell membranes permeable to DNA

Heat Shock:

• Use CaCl2 to stabalize the -DNA Phosphate Backbone

• Process of Heat Shock:

  • Ice for 10-15 minutes

  • Heat in waterbath for 90 seconds

  • Ice for 2 minutes

• Chemically competent cells uptake DNA after heat shock

Purpose of Steps:

• CaCl2

  • Transformation Solution

  • Positive charge of Ca++ ions shield negative charge of DNA phosphates

• Incubation on Ice

  • Slows fluid Cell Membrane

• Heat Shock

  • Increases Permeability of Membrane

• Nutrient Broth Incubation

  • Allows Beta-lactamase expression

  • We use Luria-Bertani(LB) Broth

    • Media that contains nutrients for bacterial growth & gene expression

      • carbohydrates

      • amino acids

      • salts

      • vitamins

Transformation Efficeny:

• percent of bacterial colonies that uptake the recombinant plasmid

• On average, only 1 in 10,000 bacterial cells take up the plasmid

• expressed as the number of transformed colonies per microgram of plasmid DNA

Steps to finding Transformation Efficency:

1. Find total mass of pGreen used in experiment

   1. Concentration of plasmid * Volume of plasmid = Total Mass of Plasmid

2. Then find total volume of cell suspension prepared

   1. CaCl2(250 uL) + LB(250 uL) + plasmid(10 uL) = Total Volume of Cell Suspension(510 uL)

3. Then find fraction of the total volume that used

   1. Volume of Cell Suspension Spread/Total volume of cell suspension that we made

4. Then find the total mass of pGreen spread on plate

   1. total mass of pGreen * Fraction spread = Mass of pGreen on plate

5. Now you can find the transformation efficency

   1. transformation efficency = # of colonies counted/mass of pGreen plasmid spread

Factors Affecting Bacterial Transformation Efficiency:

1. Competence of cells:

   1. Different bacterial strains have varying natural abilities to take up DNA. Some may require chemical treatment to become competent.

2. DNA quality:

   1. The purity, size, and concentration of the DNA being introduced can influence transformation efficiency.

3. Heat shock conditions:

   1. The duration and temperature of the heat shock step can affect the success of transformation.

4. Selection and screening:

   1. Antibiotic resistance markers or fluorescent proteins are often used to select and screen for successfully transformed cells.

Satellite Colonies: small bacterial colonies that can surround a large antibiotic‐resistant colony when grown on nutrient agar plates

• non-transformed

• Grow as the large colony secretes β-lactamase enzyme to degrade ampicillin. The degradation of ampicillin around the large colony causes the reduction of ampicillin level in that area.

https://knowt.com/note/df094e3a-cf87-4c29-88e1-2ff2ea6f99f4/Bacterial-Transformation---Research---AI