Bacterial Transformation - Research - My notes

The process by which DNA is transcribed into RNA and then translated into protein.

Green Fluorescent Protein, a protein found in Pacific Jellyfish that emits green light when excited by UV light.

The uptake of foreign DNA, often a circular plasmid, by host cells such as bacteria or yeast.

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

A circular piece of autonomously replicating DNA that can carry genes, including antibiotic resistance genes or genes of interest.

The process by which a gene is transcribed into RNA and translated into a protein.

A method that uses electrical shock to make cell membranes permeable to DNA.

A process that involves subjecting cells to heat and then cooling them rapidly, which increases the permeability of the cell membrane to DNA.

Calcium chloride, used to stabilize the DNA phosphate backbone during heat shock(Positive charge of Ca++ ions shield negative charge of DNA phosphates), transformation solution

Incubation in a nutrient-rich medium, such as Luria-Bertani (LB) broth, to allow for bacterial growth and gene expression.(Allows Beta-lactamase expression)

The percentage of bacterial colonies that successfully uptake the recombinant plasmid, expressed as the number of transformed colonies per microgram of plasmid DNA.

Only transforms 1/10,000 cells

Beta Lactamase(Ampicillin resistance)

regulator protein[(Regulates GFP transcription)(Activates under presence of arabinose which is a sugar)]

1. Ice for 10-15 minutes

2. Heat in waterbath for 90 seconds

3. Ice for 2 minutes

Once the cell membrane is more permeable, it allows for the efficient introduction of exogenous DNA through process of Heat Shock

Cells that have been treated to increase their ability to uptake foreign DNA.

Slows fluid Cell Membrane

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

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

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

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

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

small bacterial colonies that can surround a large antibiotic‐resistant colony when grown on nutrient agar plates(non-transformed)

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.

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

2. DNA quality(The purity, size, and concentration of the DNA being introduced can influence transformation efficiency)

3. Heat shock conditions(The duration and temperature of the heat shock step can affect the success of transformation)

4. Selection and screening(Antibiotic resistance markers or fluorescent proteins are often used to select and screen for successfully transformed cells)