Principles 1-3 Flashcards
what is genetic complementation
looking for genes in a pathogen that confer a virulence property on that pathogen
what do restriction enzymes do
cleave circular DNA from bacteria into smaller pieces
what are the sticky ends ligated with after it is cut by restriction enzymes
plasmid
what antibiotic kills E coli
gentamycin
gentamycin does not penetrate ____
mammalian cells
what are the different types of transposons
simple transposons and composite transposons
what does insertions of a transposon in a gene most often create
a loss of function mutation
what does a transposon mark
the site of the mutation
what are genes in simple transposons used for
transposition
what are genes in composite transposons used for
drug resistance
what happens in Tn-phoA mutagenesis
-many types of engineered transposons
- introduce Tn-phoA on a suicide plasmid
- select for KMr and screen for blue colonies
- measure PhoA activity after growth in liquid medium
- test virulence in mouse model
- result is decreased virulence
what does the phoA gene encode for
a periplasmic phosphatase mostly in gram negative cells
what does the expression of phoA depend on and why
depends on fusion to an adjacent gene after transposition because it lacks an N-terminus
what does PhoA + colonies =?
it turns blue
what osmolarity and pH are vibrio cholerae virulence genes maximally expressed at
pH of 6.5 and high osmolarity