Transformation (Day 4)

What is bacterial transformation?

Bacterial transformation is the process of introducing foreign plasmid DNA into bacterial cells. The bacteria take up the plasmid and express the genes it carries, such as an antibiotic resistance gene or a recombinant protein.

Why are competent cells required for transformation?

Competent cells have been chemically treated so they can take up plasmid DNA from their surroundings. Without competency, DNA cannot efficiently enter the bacterial cell.

Why is CaCl₂ used to make competent cells?

Both DNA and the bacterial cell surface are negatively charged, causing electrostatic repulsion. Calcium ions (Ca²⁺) neutralize these negative charges, allowing the DNA to bind to the cell surface.

What is meant by a competent cell?

A competent cell is a bacterial cell that has been treated to increase its ability to take up foreign DNA.

Why are competent cells kept on ice?

Keeping cells on ice stabilizes the cell membrane and allows plasmid DNA to bind to the cell surface before heat shock.

Why is plasmid DNA added before heat shock?

The plasmid DNA must first attach to the bacterial cell surface so it can enter the cell when temporary pores form during heat shock.

What is the purpose of heat shock?

Heat shock briefly raises the temperature to 42°C, creating temporary pores in the bacterial membrane through which plasmid DNA enters the cell.

Why is heat shock performed at 42°C?

42°C is high enough to temporarily increase membrane permeability without permanently damaging the bacterial cells.

How long is heat shock usually performed?

Heat shock is typically performed for about 30–60 seconds to maximize DNA uptake while minimizing cell damage.

What is the purpose of cold shock after heat shock?

Returning the cells to ice rapidly closes the membrane pores, trapping the plasmid DNA inside the bacterial cells.

Why is SOC medium added after transformation?

SOC is a rich recovery medium that allows transformed bacteria to repair their membranes and begin expressing the antibiotic resistance gene.

Why is SOC medium free of antibiotics?

The transformed cells need time to express the antibiotic resistance protein before being exposed to antibiotics.

Why are transformed cells incubated for about one hour before plating?

The recovery period allows the antibiotic resistance gene to be transcribed and translated so the cells can survive on selective media.

Why are transformed cells plated on antibiotic-containing agar?

Only bacteria that successfully received the plasmid will contain the antibiotic resistance gene and therefore survive on the selective agar.

What happens to bacteria that fail to take up the plasmid?

They lack the antibiotic resistance gene and are killed by the antibiotic present in the agar.

Why does the plasmid contain an antibiotic resistance gene?

It serves as a selectable marker, allowing researchers to identify bacteria that have successfully taken up the plasmid.

What is transformation efficiency?

Transformation efficiency measures how effectively bacteria take up plasmid DNA. It is expressed as colony-forming units (CFU) per microgram of plasmid DNA.

What is the formula for transformation efficiency?

Transformation Efficiency = CFU ÷ µg of plasmid DNA plated.

Why must the amount of plasmid DNA plated be calculated accurately?

Transformation efficiency is based on the amount of DNA actually spread onto the agar plate, not the total amount added to the transformation mixture.

How do you calculate the amount of plasmid DNA plated?

Calculate the total DNA in the reaction, divide by the total reaction volume to obtain DNA concentration, then multiply by the volume plated.

What is a colony-forming unit (CFU)?

A CFU represents one viable bacterial cell, or a group of cells, capable of forming a visible colony on an agar plate.

Why is JM109 commonly used during cloning?

JM109 is optimized for plasmid replication and stable maintenance of cloned DNA.

Why is BL21(DE3) not usually used for cloning?

BL21(DE3) is designed for protein expression rather than plasmid maintenance and cloning.

What makes BL21(DE3) suitable for protein expression?

It lacks the Lon and OmpT proteases, reducing degradation of recombinant proteins, and contains T7 RNA polymerase for high-level expression.

What does the "DE3" designation mean?

It indicates that the strain carries a λDE3 lysogen containing the T7 RNA polymerase gene under the control of the lacUV5 promoter.

What does "Star" mean in BL21(DE3) Star?

The strain carries an rne131 mutation that inactivates RNase E, increasing mRNA stability and improving protein production.

Why is increased mRNA stability beneficial?

Stable mRNA remains available for translation longer, allowing more recombinant protein to be produced.

What is the role of the T7 RNA polymerase?

T7 RNA polymerase recognizes the T7 promoter on the plasmid and rapidly transcribes the target gene, producing high levels of mRNA.

How does IPTG induce protein expression?

IPTG binds to the LacI repressor, causing it to detach from the operator. This allows transcription of T7 RNA polymerase, which then transcribes the target gene.

Why is IPTG preferred over lactose?

IPTG is a gratuitous inducer that is not broken down by β-galactosidase, allowing continuous induction of gene expression.

What is the role of the lac operon during protein expression?

The lac operon controls expression of T7 RNA polymerase. When IPTG removes the LacI repressor, transcription begins and the target protein is produced.