Principles 1-3 Flashcards

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1
Q

what is genetic complementation

A

looking for genes in a pathogen that confer a virulence property on that pathogen

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2
Q

what do restriction enzymes do

A

cleave circular DNA from bacteria into smaller pieces

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3
Q

what are the sticky ends ligated with after it is cut by restriction enzymes

A

plasmid

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4
Q

what antibiotic kills E coli

A

gentamycin

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5
Q

gentamycin does not penetrate ____

A

mammalian cells

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6
Q

what are the different types of transposons

A

simple transposons and composite transposons

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7
Q

what does insertions of a transposon in a gene most often create

A

a loss of function mutation

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8
Q

what does a transposon mark

A

the site of the mutation

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9
Q

what are genes in simple transposons used for

A

transposition

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10
Q

what are genes in composite transposons used for

A

drug resistance

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11
Q

what happens in Tn-phoA mutagenesis

A

-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

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12
Q

what does the phoA gene encode for

A

a periplasmic phosphatase mostly in gram negative cells

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13
Q

what does the expression of phoA depend on and why

A

depends on fusion to an adjacent gene after transposition because it lacks an N-terminus

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14
Q

what does PhoA + colonies =?

A

it turns blue

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15
Q

what osmolarity and pH are vibrio cholerae virulence genes maximally expressed at

A

pH of 6.5 and high osmolarity

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16
Q

what is the difference between genetic screen and genetic selection

A
  • genetic screen examines individual bacteria for desirable trait
  • genetic selection is where only bacteria with desirable trait grow
17
Q

describe signature tagged mutagenesis

A
  • 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
18
Q

what is promotor trapping

A

looking for genes that are expressed in infection but not in the lab
- in vitro

19
Q

what happens in DFI (differential fluorescence induction)

A

-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

20
Q

what is IVIAT ( in vivo induced antigen technology)

A
  • 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
21
Q

what are microarrays used for

A

to compare lab vs patient cases or in vivo vs in vitro

22
Q

what is whole genome sequencing used for

A

to compare nonpathogenic strains of a bacterial species to pathogenic variants

23
Q

which is more sensitive: RNA sequencing or microarray analysis

A

RNA sequencing

24
Q

which has a wider dynamic range: RNA sequencing or microarray analysis

A

RNA sequencing