Ex 1: Principles 1-3 Genetic approaches to studying pathogenic bacteria

Question 1

What is genetic complementation?

Answer 1

testing if you can create the same trait/virulence factor into one organism to another

ex: Identify Yersinia pseudotuberculosis genes that confer invasiveness on E. coli

Question 2

What is the goal for studying bacterial pathogenesis genetically?

Answer 2

to identify bacterial genes encoding virulence factors in order to develop new and better ways of preventing or treating infections

Question 3

What is the general scheme used to identify invasin (or a gene you are trying to test)?

Answer 3

1. isolate DNA and cut
2. splice donor DNA into plasmid
3. introduce into recipient
4. enrich for invaive clones
5. sequence

Question 4

How do you create the plasmids you want to test to transform into E coli?

Answer 4

• digest genome of og bacteria with restriction endonuclease into restriction fragments
• ligate with plasmid digested to generate sticky ends
• plasmids with DNA and cloned donor DNA

Question 5

What type of selection is used to test that the E coli conferred the invasion gene?

Answer 5

positive selection (pick and test individually)

Question 6

What does gentamycin penetrate?

Answer 6

kills the E coli that do not invade mammalian cells

• does not penetrate mammalian cells

Question 7

What are the steps to test E coli invasion ability?

Answer 7

1. grow in culture
2. inoculate microtiter wells
3. incubate
4. wash
5. gentamicin-containing medium
6. gentle lysis
7. titer for viable counts

Question 8

What does a suicide plasmid mean?

Answer 8

• the plasmid cannot replicate in the og bacteria
• it can replicate in E coli

Question 9

What is required for the plasmid to get inserted into the bacterial chromosome?

Answer 9

double recombination

Question 10

How many recombination events are needed to replace the inv gene with the loss-of-function allele?

Answer 10

2

Question 11

What are the parts of the simple transposon?

Answer 11

core area
inverted repeat sequences

Question 12

What are the parts of the composite transposon?

Answer 12

core area
terminal IS elements

Question 13

Insertion of a transposon in a gene most often creates a _________ mutation

Answer 13

loss-of-function

Question 14

Transposon marks the site of the _______

Answer 14

mutation

Question 15

What does the phoA gene encode?

Answer 15

encodes a periplasmic phosphatase
- lacks N-terminus

Question 16

What does phoA gene lack and what does that do?

Answer 16

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

Question 17

What happens to the PhoA+ colonies

Answer 17

They turn blue (cut X-P dye)

Question 18

What does Tn-phoA mutagenesis allow for?

Answer 18

testing for decreased virulence

Question 19

What does selection mean?

Answer 19

only bacteria with desirable trait grow

Question 20

What does screen mean?

Answer 20

examine individual bacteria for
desirable trait

Question 21

Does signature-tagged mutatgenesis screen or select for traits?

Answer 21

screens for negatie traits

Question 22

What do the dark circles versus the blank spots mean on the recovered pool blot?

Answer 22

• dark circles are the bacteria that survived in the mouse
• blank spot represent bacteria that did not survive in the mouse

Question 23

What does promoter-trapping (IVET: in vitro expression technology) allow for?

Answer 23

look for genes that are expressed in infection but not in the laboratory

Question 24

What is the LacZY genes function in the plasmid?

Answer 24

show which genes are Lac- in vitro and only grow in vivo

Question 25

What is DFI: differential fluorscence induction?

Answer 25

used to identify genes expressed in bacteria during intracellular survival in macrophages

Question 26

What is IVIAT: in vivo-induced antigen technology?

Answer 26

antibody-based approach used to identify in vivo-induced antigens

Question 27

What is the point of a microarray?

Answer 27

used to compare the transcriptomes of certain bacteria in vitro versus in vivo

Question 28

What would yellow, red, and green signals mean?

Answer 28

yellow signal = both bacterial genomes are expressed equal

red signal = more RNA expressed (gene expression) in lab sample

green signal = more RNA expressed (gene expression) in disease setting

Question 29

What is the purpose of using whole genome sequencing?

Answer 29

to compare nonpathogenic strains of a bacterial species to a pathogenic variant (compare the regions that differ)

Question 30

What is the difference between total RNA sequencing verus microarry analysis?

Answer 30

• microarray uses analogue quantification
• RNA sequencing is more sensitive and has a wider dynamic range than microarray analysis (digital quantification)

Question 31

What is different in the processing between microarray and RNA sequencing?

Answer 31

• microarray you use physical separation
• RNA sequecing you use collective processing and do not need to separate host versus pathogen