1-3 part A, B, C Flashcards
insertion of a transposon in a gene most often creates a
loss of function mutation
transposon marks the site of:
(sequences and antibiotic resistance)
mutation
insertion sequence (IS) element
simple transposon
contains two IS elements and genes for drug resistance
composite transposon
encodes a periplasmic phosphatase
phoA gene
engineer phoA gene lacks N-terminus so expression depends on fusion to
an adjacent gene after transpotion
vibrio cholerae virulence genes maximally expressed at pH
6.5 and high osmolarity
examine individual bacteria for desirable trait
screen
(example: just the blue colonies vs. white colonies)
only bacteria with desirable trait grow (gentamycin selection in complementation assay)
selection
(example: all the colonies that contain Kmr gene or transposons)
this is a type of screen for a negative trait: inability to grow in spleen
- looking for loss-of-function recovered results which are important for survival in a mouse
- we are finding genes that are necessary for the survival in a host and NOT in lab
2.
signature-tagged mutagenesis
(studying mouse model for typhoid fever)
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)
signature-tagged mutagensis
in this type of study, we look for genes of S. typhimurium that are expressed in infection, but NOT in the laboratory
promotor-trapping
(IVET: in vitro expression technology)
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.
promotor-trapping
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?!
promotor trapping
means that those bacteria without dye can help tell what genes are expressed in the mouse and not in the lab
(cell sorting)
uses ‘gfp to identify then infect macrophages and separate infected macrophages via FACS
DFI: differential fluorescence induction
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?
- 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
- 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.
what is the purpose of the differential fluorescence induction
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)
this study is an antibody-based approach
IVIAT: in vivo-induced antigen technology
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
in vivo-induced antigen technology
what is the purpose of in vivo-induced antigen technology
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
this is used to compare lab grown bacteria (in vitro) and a patient’s sample (in vivo) and see which is more
transcription analysis 1: microarrays
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
genome analysis: whole genome sequencing (DNA based approach)
this is used to find virulence factor by comparing whole genome of non-pathogenic strains to whole genome sequence of pathogen variants
whole genome sequencing
total RNA sequencing of bacterial samples compared to microarray analysis of bacterial RNA
transcription analysis 2: RNA sequencing
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
RNA sequencing
this is the sequencing of a total RNA population of the pathogeon
RNA sequencing (when compared to microarray)
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
microarry
microarray vs rna sequencing, which is this?
you collect everything together and sequence all RNA that you isolate from the host RNA
RNA sequencing (easier)
collective process
