Principles 1-3

Question 1

what is genetic complementation

Answer 1

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

Question 2

what do restriction enzymes do

Answer 2

cleave circular DNA from bacteria into smaller pieces

Question 3

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

Answer 3

plasmid

Question 4

what antibiotic kills E coli

Answer 4

gentamycin

Question 5

gentamycin does not penetrate ____

Answer 5

mammalian cells

Question 6

what are the different types of transposons

Answer 6

simple transposons and composite transposons

Question 7

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

Answer 7

a loss of function mutation

Question 8

what does a transposon mark

Answer 8

the site of the mutation

Question 9

what are genes in simple transposons used for

Answer 9

transposition

Question 10

what are genes in composite transposons used for

Answer 10

drug resistance

Question 11

what happens in Tn-phoA mutagenesis

Answer 11

-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

Question 12

what does the phoA gene encode for

Answer 12

a periplasmic phosphatase mostly in gram negative cells

Question 13

what does the expression of phoA depend on and why

Answer 13

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

Question 14

what does PhoA + colonies =?

Answer 14

it turns blue

Question 15

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

Answer 15

pH of 6.5 and high osmolarity

Question 16

what is the difference between genetic screen and genetic selection

Answer 16

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

Question 17

describe signature tagged mutagenesis

Answer 17

• 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

Question 18

what is promotor trapping

Answer 18

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

Question 19

what happens in DFI (differential fluorescence induction)

Answer 19

-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

Question 20

what is IVIAT ( in vivo induced antigen technology)

Answer 20

• 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

Question 21

what are microarrays used for

Answer 21

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

Question 22

what is whole genome sequencing used for

Answer 22

to compare nonpathogenic strains of a bacterial species to pathogenic variants

Question 23

which is more sensitive: RNA sequencing or microarray analysis

Answer 23

RNA sequencing

Question 24

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

Answer 24

RNA sequencing