Microbial Genetics

What is genetics?

The study of genes, heredity, and genetic variation in organisms.

What is a mutation?

A permanent change in the DNA sequence.

What causes mutations?

Mutagens such as UV radiation, chemicals, or errors during DNA replication.

What is a mutagen?

An agent that increases the frequency of mutations in DNA.

What is a prototroph?

A microorganism that can synthesize all essential nutrients needed for growth.

What is an auxotroph?

A mutant microorganism that has lost the ability to synthesize one or more essential nutrients.

What nutrient must an auxotroph receive?

The specific nutrient it can no longer synthesize.

How can a prototroph grow on minimal media?

It synthesizes all required nutrients on its own.

Why can't an auxotroph grow on minimal media?

It lacks the ability to make an essential nutrient.

What technique is used to identify auxotrophs?

Replica plating.

What is replica plating?

A technique that transfers the same bacterial colony pattern onto multiple agar plates.

Why are reference marks placed on the bottom of plates before replica plating?

To ensure colonies can be matched between plates.

What was the purpose of replica plating in this lab?

To identify mutant colonies that require nutrients missing from minimal media.

Which two media were compared during replica plating?

TSA and glucose-minimal salts agar.

Why can prototrophs grow on both TSA and minimal salts agar?

They synthesize all essential nutrients.

Why do auxotrophs grow on TSA but not minimal salts agar?

TSA contains nutrients they cannot synthesize.

What indicates that a colony is an auxotroph?

It is present on TSA but absent from the corresponding location on minimal salts agar.

What organism was used during the replica plating experiment?

Serratia.

Why was Serratia incubated at 25°C?

It grows well at that temperature and produces characteristic pigmentation.

What is the purpose of serial dilution before plating?

To reduce bacterial concentration and produce isolated colonies.

Why are isolated colonies important?

They allow individual mutants to be identified and transferred.

What is an operon?

A group of genes regulated together under the control of a single promoter.

What is the ara operon?

An inducible operon that controls the metabolism of arabinose.

What sugar activates the ara operon?

Arabinose.

What does inducible mean?

A gene system that is normally OFF but can be turned ON when a specific molecule is present.

When is the ara operon turned ON?

When arabinose is present.

When is the ara operon turned OFF?

When arabinose is absent.

What plasmid was used in the ara operon experiment?

pGLO.

What does the pGLO plasmid contain?

The GFP gene under control of the ara operon.

What does GFP stand for?

Green Fluorescent Protein.

Where does the GFP gene originally come from?

The jellyfish Aequorea victoria.

What causes GFP to fluoresce?

Exposure to ultraviolet light after expression of the GFP protein.

Why do pGLO colonies glow only on LB + arabinose plates?

Arabinose activates the ara operon, allowing GFP to be produced.

Why don't pGLO colonies glow on regular LB plates?

Without arabinose, the ara operon remains OFF and GFP is not produced.

What medium was used for the pGLO experiment?

LB agar and LB agar containing arabinose.

What does LB stand for?

Luria-Bertani broth or agar, a nutrient-rich medium.

What was the purpose of comparing LB and LB + arabinose plates?

To demonstrate regulation of gene expression by the ara operon.

What color do colonies appear under UV light when GFP is expressed?

Bright green.

Does UV light activate the ara operon?

No. UV only allows visualization of GFP after it has been produced.

What molecule actually activates GFP production?

Arabinose.

What is gene regulation?

The control of when genes are turned ON or OFF.

Why is the ara operon considered inducible?

It requires arabinose to activate gene transcription.

What is transcription?

The process of copying DNA into messenger RNA.

What is translation?

The process of using mRNA to produce proteins.

What protein is produced after activation of the ara operon in pGLO?

Green Fluorescent Protein (GFP).

Why is GFP useful in research?

It serves as a visible marker of gene expression.

What was observed under the UV lamp during the pGLO experiment?

Colonies on LB + arabinose fluoresced green, while colonies on LB did not.

What is the purpose of minimal salts agar?

To determine whether bacteria can synthesize essential nutrients.

What conclusion can be made if a colony grows on TSA but not minimal salts agar?

The colony is an auxotroph.

What conclusion can be made if a colony grows on both TSA and minimal salts agar?

The colony is a prototroph.

Why are mutations important in genetics?

They create genetic variation and may alter cellular functions.

How can UV light produce auxotrophs?

UV radiation induces DNA mutations that may disrupt nutrient synthesis pathways.

What was the overall goal of the microbial genetics labs?

To understand mutations, gene regulation, operons, GFP expression, and identification of auxotrophic mutants.

What is the difference between a prototroph and an auxotroph?

A prototroph synthesizes all required nutrients; an auxotroph requires a specific nutrient supplied in the medium.

What is the difference between LB agar and minimal salts agar?

LB is nutrient-rich, while minimal salts agar contains only essential nutrients and requires organisms to synthesize everything else.

What is the difference between an inducible operon and a repressible operon?

An inducible operon is normally OFF and turned ON by an inducer, while a repressible operon is normally ON and turned OFF by a corepressor.