1.3

Question 1

creating a genomic library

Answer 1

genome of the organism (chromosomal DNA)
digest with a restriction endonuclease 
restriction fragments 
ligate with plasmid digested to generate the same "sticky ends"
DNA with cloned donor DNA

Question 2

— kills
E. coli that do not
invade.

Answer 2

Gentamycin

Question 3

Gentamycin does
not penetrate
— cells.

Answer 3

mammalian

Question 4

Positive selection

Answer 4

grow in culture 
inoculate microfilter wells 
incubate 37C
gentamicin-containing medium
gentile lysis 
titer for viable counts 
pick and test individually

Question 5

After identification of

gene for invasion factor:

Answer 5

Generate DNA sequence
= inv gene
Deduce protein coding region
= invasin protein

Question 6

Manipulate gene further to
prove that invasin really
does promote

Answer 6

cell invasion.

Question 7

Plasmid cannot replicate in
— (but it
can replicate in E. coli)

Answer 7

Y. pseudotuberculosis

Question 8

a majority of the inv gene contained on a

Answer 8

suicide plasmid

Question 9

Suicide plasmid containing inv loss-of-function mutation transferred
from

Answer 9

E. coli to Y. pseudotuberculosis

Question 10

Need 2 recombination events to

Answer 10

replace the inv gene in Y. pseudotuberculosis

with the loss-of-function inv allele

Question 11

Test Y. pseudotuberculosis inv mutants and show that

Answer 11

they do not invade.

Question 12

Insertion of a transposon in a gene most often creates a

Answer 12

loss-of-function mutation.

Question 13

Transposon marks the site of the

Answer 13

mutation (sequence and antibiotic resistance)

Question 14

phoA gene

Encodes a

Answer 14

periplasmic

phosphatase

Question 15

engineered phoA gene
lacks N
-terminus so
expression depends
on

Answer 15

fusion to an
adjacent gene after
transposition

Question 16

PhoA+ colonies turn

Answer 16

blue (cut X

-P dye)

Question 17

Vibrio cholerae
virulence genes
maximally
expressed at pH
--- and high
---

Answer 17

6.5

osmolarity

Question 18

result?

Answer 18

Decreased virulence

Question 19

Tn-phoA mutagenesis

Answer 19

introduce tn-phoA on a suicide plasmid
select for Km and screen for blue colonies
measure phoA activity after growth in liquid medium
test virulence in mouse model (decreased virulence)

Question 20

screen

Answer 20

examine individual bacteria for

desirable trait

Question 21

selection

Answer 21

only bacteria with desirable trait grow
(Gentamycin selection in
complementation assay)

Question 22

Signature-tagged mutagenesis
This is a — for a
negative trait: inability to
grow in spleen.

Answer 22

screen

Question 23

Studying — model for typhoid fever

Answer 23

mouse

Question 24

Signature-tagged mutagenesis

Answer 24

Unique marker put
into each transposon
Create a library of S.
typhimurium mutants
that each contain a
mini-Tn5 insertion
(suicide plasmid used to
move mini-Tn5 from E.
coli into S. typhimurium)

Question 25

Promoter-trapping (IVET: in vitro expression technology)

Answer 25

``` Look for genes of S. typhimurium that are expressed in infection but not in the laboratory This is only one strategy but it is doable. ```

Question 26

Promoter-trapping (IVET: in vitro expression technology)

Answer 26

partial digestion of salmonella chromosome ligate and propagate in e coli conjugal transfer to a salmonella purine synthesis mutant integration of the plasmid onto the chromosomes inoculation of a pool of fusions, incubate x3 days to select pur strain in vivo recover bacteria from spleen screen for lac- invitro

Question 27

DFI: Differential fluorescence induction

Answer 27

partial digestion of salmonella chromosome ligate into a gfp-fusion plasmid an transform salmonella infect macrophages with pooled, transferred salmonella and separate them by FACs lyse macrophages with fluorescent bacteria, grow on media, 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 28

IVIAT: In vivo-induced antigen technology (Antibody-based | approach)

Answer 28

create an expression library replicate library after induction with IPTG remove ab 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 ab reactive proteins

Question 29

Transcription analysis 1:

Answer 29

microarrays

Question 30

Genome analysis:

Answer 30

whole genome sequencing (DNA based approach)

Question 31

Whole genome sequencing to compare

Answer 31

nonpathogenic strains of a bacterial species to pathogenic variants

Question 32

Transcription | analysis 2:

Answer 32

RNA sequencing

Question 33

Total RNA sequencing of bacterial samples compared to

Answer 33

microarray analysis of bacterial RNA

Question 34

RNA sequencing

Answer 34

= sequencing of a total RNA | population of the pathogen

Question 35

RNA sequencing is more --- and has a --- dynamic range than microarray analysis.

Answer 35

sensitive | wider