Screens, selections and enrichments Flashcards
How is an enrichment different from a selection?
Enrichments increase the proportion of mutants in the population, while a selection just kills anything that isn’t a mutant
What does penicillin do?
Blocks the formation of cross links in PG and causes the cell wall to be really weak. But it only affects growing cells
How do you do a penicillin enrichment?
- Mutagenesis
- Grow everything in permissive conditions
- Transfer to restrictive conditions
- Add penicillin
- Osmotic shock
- Plate survivors in permissive conditions
- Repeat a few times then recover the mutants
What is a screen?
Everything grows, but the mutant has a distinct phenotype from the WT
Why do we need to do replica plating?
To screen for conditional lethal mutants because we need to compare different conditions
How do you do replica plating?
- Plate the cells under permissive conditions
- Make a copy of the plate with velveteen or toothpicks
- Put one plate at restrictive conditions
- Look for stuff that grows on one but not the other plate
What is a selection?
Only things that grow are your mutants of interest. Everything else dies
If selections are easier than screening, why don’t we do them all the time?
Most mutations can’t be selected for, since mutations usually cause the cell to grow worse than the WT, so we have to screen for those
What sort of mutations are really easy to select for?
Antibiotic resistance or auxotrophic revertants
Why would we do an enrichment?
Increases the proportion of mutants in the population, so you have less to screen to find a mutant
