lec 4

Flashcards covering key vocabulary, laboratory workflows, and analytical strategies for bacterial mutagenesis, counter-selection enrichments, and genetic complementation assays.

Mutagenesis reaction considerations

The three physical and chemical factors controlling a mutagenesis experiment: (1) chemical reactivity of the mutagen with DNA, (2) concentration of both the mutagen and target DNA, and (3) amount of time that DNA is exposed to the mutagen.

Heavy mutagenesis

An intense mutagenesis procedure used when desired mutations are expected to be rare; it is characterized by substantial cell death and can be monitored by observing elevated cell lethality or measuring mutation rates.

• used when a mutation is rare

Screen or Select for: Isolate mutations in a gene encoding a protein that provides resistance to an antibiotic

SCREEN

• SCREEN b/c youre starting with a proteins that has a resistance function . therefore you want to screen for the mutation with a loss of function to the resistance function

Screening vs. selection rule of thumb

The general principle that loss-of-function mutations must typically be isolated by screening (inspecting all individuals), whereas gain-of-function mutations can generally be isolated by selection (establishing conditions where only mutants grow).

Enrichment

A technique applied to rare mutations where mutant cells cannot grow under selective conditions (grow/no-grow screening situations); it uses conditions that selectively kill actively growing parent cells to enhance the proportion of surviving mutant cells in the population.

• used when mutation is rare and doesnt grow under SELECTIVE conditions

Ampicillin enrichment

A transient liquid culture procedure where ampicillin kills actively growing and dividing parental cells by targeting peptidoglycan synthesis, while non-growing mutant cells survive and are subsequently rescued by transfer to nonselective media.

Blue-white screening non-enrichability

The inability to perform enrichment on $lacZ$ mutants cultured with IPTG and X-Gal because both parental and mutant cells survive and form colonies (differing only by color) rather than displaying a growth versus no-growth phenotype.

Genome sequencing (in mutant analysis)

The method utilized following mutagenesis and screening/selection to pinpoint the exact sequence and chromosomal location of the mutation causing a phenotype.

Complementation

The genetic technique of introducing an intact wild-type copy of a gene into a mutant strain to determine whether it restores the wild-type phenotype, thereby proving whether that specific mutation caused the observed phenotype.

Complementation workflow

The molecular protocol of isolating chromosomal DNA from a wild-type strain, using flanking primers to PCR-amplify the wild-type gene, cloning it into a plasmid, and transforming the construct into the mutant cells.

Plasmid selection in complementation

The use of an antibiotic resistance marker encoded on the complementation plasmid to select for bacteria that have successfully taken up the plasmid by plating them on antibiotic-containing media.

Complementation selection of loss-of-function mutants

The direct selection for complemented strains on selective media (such as media lacking an amino acid like cysteine or tryptophan, or at restrictive temperatures) where only cells receiving the functional wild-type allele regain the ability to grow.

Complementation limitation on selected mutations

The incompatibility of testing complementation under the original selective conditions for mutations that conferred survival, because restoring the wild-type allele eliminates the selected survival trait and causes cell death under those selective conditions.