Lab 3 Flashcards

Transformation

Uptake and incorporation of naked DNA from the environment into a competent bacterial cell.

Transduction

Transfer of bacterial DNA via a bacteriophage.

Conjugation

One‑way transfer of DNA between two bacterial cells through a pilus.

Gene transfer (bacteria)

Movement of genetic material between bacterial cells through transformation, transduction, or conjugation.

Competence

A physiological state in which a bacterial cell can take up external DNA.

Griffith experiment

An experiment showing that non‑virulent rough S. pneumoniae became virulent when mixed with heat‑killed smooth cells due to DNA uptake (transformation).

Smooth strain (S)

Virulent strain of S. pneumoniae that kills mice.

Rough strain (R)

Non‑virulent strain of S. pneumoniae that does not kill mice.

Heat‑killed smooth strain

Non‑virulent; cannot kill mice but can donate DNA.

Why rough + heat‑killed smooth kills mice

Live rough cells take up DNA from dead smooth cells and become virulent.

Plasmid

Small circular DNA molecule that replicates independently of the bacterial chromosome.

pBLU plasmid

A plasmid containing amp^R and lacZ genes used for transformation experiments.

amp^R

Gene that provides resistance to ampicillin.

lacZ

Gene encoding β‑galactosidase, which digests lactose and turns colonies blue on X‑gal.

β‑galactosidase

Enzyme that cleaves lactose and X‑gal, producing blue colonies.

Selectable marker

A gene that allows identification of transformed cells (e.g., antibiotic resistance).

Blue‑white screening

A method using X‑gal to detect lacZ activity; blue colonies indicate lacZ expression.

LB plate

A nutrient medium that supports bacterial growth; used to check viability.

LB/amp plate

A selective medium containing ampicillin; only transformed cells with amp^R grow.

LB/X‑gal plate

A medium containing X‑gal; lacZ‑expressing cells turn blue.

LB/amp/X‑gal plate

A selective and differential medium; only transformed cells grow, and lacZ expression produces blue colonies.

Expected result on LB (+ plasmid)

Growth (cells are alive).

Expected result on LB (– plasmid)

Growth (cells are alive).

Expected result on LB/amp (+ plasmid)

Growth due to amp^R.

Expected result on LB/amp (– plasmid)

No growth (no amp^R).

Expected result on LB/X‑gal (+ plasmid)

Blue colonies (lacZ expressed).

Expected result on LB/X‑gal (– plasmid)

White colonies (no lacZ).

Expected result on LB/amp/X‑gal (+ plasmid)

Blue colonies (amp^R + lacZ).

Expected result on LB/amp/X‑gal (– plasmid)

No growth.

How to know transformation succeeded

Cells grow on ampicillin plates and produce blue colonies on X‑gal.

Ori (origin of replication)

Region of plasmid that allows independent replication inside bacteria.

MCS (multiple cloning site)

Region of plasmid containing many restriction sites for inserting DNA.

E. coli DH5α

A laboratory strain of E. coli used for transformation due to high competence.

X‑gal

A substrate that turns blue when cleaved by β‑galactosidase.

Antibiotic resistance gene

A gene that allows bacteria to survive in the presence of an antibiotic.

Field of View (FOV)

A counted region used to quantify colony growth or compare before/after results.

Purpose of selective media

To ensure only transformed cells survive and express plasmid genes.

Purpose of X‑gal

To visually indicate lacZ activity through blue colony formation.

Why native E. coli cannot grow on ampicillin

It lacks the amp^R gene.

Why native E. coli cannot digest lactose

It lacks the lacZ gene.

Why plasmids are useful

They carry genes of interest and replicate independently, making them ideal for genetic engineering.

What transformation demonstrates about DNA

DNA is the molecule that carries heritable traits.

Rubric location

Back of lab manual pg. 72; Canvas under Lab Report 1; lab report example.