Bacterial Transformation - Research - My notes

Central Dogma

Central Dogma

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

GFP

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

Transformation

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

Transformation Principles

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

Plasmid

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

Gene Expression

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

Electroporation

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

Heat Shock

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

CaCl2

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

Nutrient Broth Incubation

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

Transformation Efficiency

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

Why is Transformation inefficent?

Only transforms 1/10,000 cells

bla

Beta Lactamase(Ampicillin resistance)

araC

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

Process of Heat Shock in Lab

1. Ice for 10-15 minutes

2. Heat in waterbath for 90 seconds

3. Ice for 2 minutes

When do competent cells take up DNA?

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

Competent Cells

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

Incubation on Ice

Slows fluid Cell Membrane

How to find total mass of pGreen used in experiment?

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

How to find total volume of cell suspension prepared?

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

How to find fraction of the total volume that used?

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

How to find the total mass of pGreen spread on plate?

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

How to find the transformation efficency?

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

Satellite Colonies

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

Why can satellite colonies grow without ampicillin resistance?

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.

Factors Affecting Bacterial Transformation Efficiency

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)