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
what is the difference between genetic screen and genetic selection
- genetic screen examines individual bacteria for desirable trait
- genetic selection is where only bacteria with desirable trait grow
describe signature tagged mutagenesis
- start with microfilter plate with tagged transposon mutants
- pool mutants
- inject mouse
- extract spleen of mouse and recover bacteria from spleen and put on agar plate
- extract DNA and amplify tags by PCR
- divided into input pool and recovered pool
what is promotor trapping
looking for genes that are expressed in infection but not in the lab
- in vitro
what happens in DFI (differential fluorescence induction)
-partial digestion of salmonella chromosome
- ligate into a gfp- fusion plasmid and transform salmonella
- infect macrophages with pooled, transformed salmonella and separate them by FACS
- lyse macrophages with fluorescent bacteria, grow on medium then sort bacteria by FACS
- infect macrophages with nonfluorescent sorted bacteria and then sort macrophages to isolate with fluorescent bacteria
- analyze cloned sequences in bacteria that are fluorescent when grown in macrophages but non fluorescent on media
what is IVIAT ( in vivo induced antigen technology)
- antibody based approach
- start with genomic DNA fragments and create a lambdagt11 expression library
- replicate library after induction with IPTG
- remove antibodies from patient serum that bind to factors from organisms grown on medium
- perform in situ immunoassay of the expression library with absorbed antiserum
- isolate phage DNA, sequence and characterize insert expressing antibody reactive proteins
what are microarrays used for
to compare lab vs patient cases or in vivo vs in vitro
what is whole genome sequencing used for
to compare nonpathogenic strains of a bacterial species to pathogenic variants
which is more sensitive: RNA sequencing or microarray analysis
RNA sequencing
which has a wider dynamic range: RNA sequencing or microarray analysis
RNA sequencing
