1-3 part A, B, C

Question 1

insertion of a transposon in a gene most often creates a

Answer 1

loss of function mutation

Question 2

transposon marks the site of:

(sequences and antibiotic resistance)

Answer 2

mutation

Question 3

insertion sequence (IS) element

Answer 3

simple transposon

Question 4

contains two IS elements and genes for drug resistance

Answer 4

composite transposon

Question 5

encodes a periplasmic phosphatase

Answer 5

phoA gene

Question 6

engineer phoA gene lacks N-terminus so expression depends on fusion to

Answer 6

an adjacent gene after transpotion

Question 7

vibrio cholerae virulence genes maximally expressed at pH

Answer 7

6.5 and high osmolarity

Question 8

examine individual bacteria for desirable trait

Answer 8

screen
(example: just the blue colonies vs. white colonies)

Question 9

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

Answer 9

selection
(example: all the colonies that contain Kmr gene or transposons)

Question 10

this is a type of screen for a negative trait: inability to grow in spleen

1. looking for loss-of-function recovered results which are important for survival in a mouse
2. we are finding genes that are necessary for the survival in a host and NOT in lab

2.

Answer 10

signature-tagged mutagenesis

(studying mouse model for typhoid fever)

Question 11

with this study, there are unique markers put into each transposon to create a library of S. typhimurium mutants that each contain a mini-Tn5 insertion.

a suicide plasmid is then used to move mini-Tn5 from E. coli into S. typhimurium)

Answer 11

signature-tagged mutagensis

Question 12

in this type of study, we look for genes of S. typhimurium that are expressed in infection, but NOT in the laboratory

Answer 12

promotor-trapping
(IVET: in vitro expression technology)

Question 13

this type of study is different from transposon insertion aka loss-of-function method because the X’ still has one intact copy of the target gene.

Answer 13

promotor-trapping

Question 14

the laxZY encodes enzyme in lactose utilization for which a dye is able to be put into a medium and is cleared when lacZY is active.

therefore, the bacteria on the well that does not have dye can’t cleave it, so it is not expressing fusion for lacZY or purA.

what type of study is this and what does this mean?!

Answer 14

promotor trapping

means that those bacteria without dye can help tell what genes are expressed in the mouse and not in the lab

Question 15

(cell sorting)
uses ‘gfp to identify then infect macrophages and separate infected macrophages via FACS

Answer 15

DFI: differential fluorescence induction

Question 16

in Differential Fluorescene Induction (DFI) once the macrophages are sorted on those that are infected with ‘gfp and those without ‘gfp, what are the two steps after this?

Answer 16

1. the macrophages that are infected with ‘gfp are then put through FACS to split between the bacteria in them with the ‘gfp and the bacteria without ‘gfp
2. then, macrophages are again infected with the bacteria without ‘gfp.
   Then the macrophages are sorted to isolate those who appear to have the active ‘gfp and those who do not have the ‘gfp.

Question 17

what is the purpose of the differential fluorescence induction

Answer 17

we are looking for bacteria that will express ‘gfp (flourescence) while in macrophages, but NOT express ‘gfp under lab conditions (aka outside of the macrophages)

Question 18

this study is an antibody-based approach

Answer 18

IVIAT: in vivo-induced antigen technology

Question 19

this study uses plaques (filled with microbacterial proteins) and takes human antibodies that have been cleared from other antibodies that can bind to other growth factors (like e.coli) and joins them to see what plaques react with the antibodies

Answer 19

in vivo-induced antigen technology

Question 20

what is the purpose of in vivo-induced antigen technology

Answer 20

to see what antibodies react with the plaques because this means they contain microbacterial proteins and may play a role in tuberculosis and pathogens of that bacterium

Question 21

this is used to compare lab grown bacteria (in vitro) and a patient’s sample (in vivo) and see which is more

Answer 21

transcription analysis 1: microarrays

Question 22

this compares nonpathogenic strains of a bacterial species to pathogenic variants.

red regions are unqiue to the strain of bacteria when the two are compared

Answer 22

genome analysis: whole genome sequencing (DNA based approach)

Question 23

this is used to find virulence factor by comparing whole genome of non-pathogenic strains to whole genome sequence of pathogen variants

Answer 23

whole genome sequencing

Question 24

total RNA sequencing of bacterial samples compared to microarray analysis of bacterial RNA

Answer 24

transcription analysis 2: RNA sequencing

Question 25

when composing the transcription analysis 2: RNA sequencing and comparing Microarray approach to the RNA-sequence approach,

which approach is more sensitive and has a wider dynamic range

Answer 25

RNA sequencing

Question 26

this is the sequencing of a total RNA population of the pathogeon

Answer 26

RNA sequencing (when compared to microarray)

Question 27

microarray vs rna sequencing, which is this?

you isolate pathogen and isolate the RNA from it so you can look at the pathogen RNA in context of infection situation

Answer 27

microarry

Question 28

microarray vs rna sequencing, which is this?

you collect everything together and sequence all RNA that you isolate from the host RNA

Answer 28

RNA sequencing (easier)

collective process