Ex 1: Principles 1-3 Genetic approaches to studying pathogenic bacteria Flashcards
What is genetic complementation?
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
What is the goal for studying bacterial pathogenesis genetically?
to identify bacterial genes encoding virulence factors in order to develop new and better ways of preventing or treating infections
What is the general scheme used to identify invasin (or a gene you are trying to test)?
- isolate DNA and cut
- splice donor DNA into plasmid
- introduce into recipient
- enrich for invaive clones
- sequence
How do you create the plasmids you want to test to transform into E coli?
- 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
What type of selection is used to test that the E coli conferred the invasion gene?
positive selection (pick and test individually)
What does gentamycin penetrate?
kills the E coli that do not invade mammalian cells
- does not penetrate mammalian cells
What are the steps to test E coli invasion ability?
- grow in culture
- inoculate microtiter wells
- incubate
- wash
- gentamicin-containing medium
- gentle lysis
- titer for viable counts
What does a suicide plasmid mean?
- the plasmid cannot replicate in the og bacteria
- it can replicate in E coli
What is required for the plasmid to get inserted into the bacterial chromosome?
double recombination
How many recombination events are needed to replace the inv gene with the loss-of-function allele?
2
What are the parts of the simple transposon?
core area
inverted repeat sequences
What are the parts of the composite transposon?
core area
terminal IS elements
Insertion of a transposon in a gene most often creates a _________ mutation
loss-of-function
Transposon marks the site of the _______
mutation
What does the phoA gene encode?
encodes a periplasmic phosphatase
- lacks N-terminus
What does phoA gene lack and what does that do?
lacks N-terminus
- expression depends on fusion to an adjacent gene after transposition
What happens to the PhoA+ colonies
They turn blue (cut X-P dye)
What does Tn-phoA mutagenesis allow for?
testing for decreased virulence
What does selection mean?
only bacteria with desirable trait grow
What does screen mean?
examine individual bacteria for
desirable trait
Does signature-tagged mutatgenesis screen or select for traits?
screens for negatie traits
What do the dark circles versus the blank spots mean on the recovered pool blot?
- dark circles are the bacteria that survived in the mouse
- blank spot represent bacteria that did not survive in the mouse
What does promoter-trapping (IVET: in vitro expression technology) allow for?
look for genes that are expressed in infection but not in the laboratory
What is the LacZY genes function in the plasmid?
show which genes are Lac- in vitro and only grow in vivo
What is DFI: differential fluorscence induction?
used to identify genes expressed in bacteria during intracellular survival in macrophages
What is IVIAT: in vivo-induced antigen technology?
antibody-based approach used to identify in vivo-induced antigens
What is the point of a microarray?
used to compare the transcriptomes of certain bacteria in vitro versus in vivo
What would yellow, red, and green signals mean?
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
What is the purpose of using whole genome sequencing?
to compare nonpathogenic strains of a bacterial species to a pathogenic variant (compare the regions that differ)
What is the difference between total RNA sequencing verus microarry analysis?
- microarray uses analogue quantification
- RNA sequencing is more sensitive and has a wider dynamic range than microarray analysis (digital quantification)
What is different in the processing between microarray and RNA sequencing?
- microarray you use physical separation
- RNA sequecing you use collective processing and do not need to separate host versus pathogen
