seminar 4 Flashcards
- What are the benefits and limits of the microarray-based method?
it can have false negatives/positives
microarray doesnt represent entire genome
different hybridization temperatures/conditions
transposon will not saturate gerne, inserted only every 1000 nucleotides, a lot of genes never mutated
- What is special about the transposon used in this microarry-based study? What was its importance for this study?
Tn10 transposon library (50,000 individual mutants) was inocuulated into mice to identify serovar typhimurium genes that contribute to the establishment and maintenance of a long-term infection in mice
each transposon insertion containt T7 transcriptional IPTG inducible promoter
in vitro transcription of genes
- What is the significance of the infection route used?
intra peritoneal
previous studies have shown that the oral route imposes a significant bottleneck in which less than 1% of bacteria were able to successfully breach the intestinal barrier and colonize systemic tissues
you maybe loose virulence genes important for oral route, but you get way more new information
IP route minimal impact, from IP bacteria can get into spleen and liver with same efficiency
- Why did the authors measure Competitive Index?
validade results
take some genes: head to head comp with wt, mutant should not outcompete wt
2. how important are they? 10% less fit 50% less fit, but mostly for validation of results
to further investigate the role of individual virulence factors that have not previously been implicated in systemic disease.
measurment of relative fitness (mutant to wild type)
(inject mouse with same amount of mutant and wild type and see which is more prevalent after 30 days (wildtype)
- What is the TraDIS method?
important!
transposon-directed insertion-site sequecing
The sequences of the regions next to transposons were sequenced/identified
permits comparison of the number of specific reads derived from the input pools and the output pools after animal infection -> numerical measure
- How was TraDIS used to identify genes essential for colonization?
mutants that grow in culture but do not survive in one or more of the animals. these genes are important for the infection process
tradis shows semi quantitive data, so not only which ones are essential, but also which ones help
9.Were there any significant differences in gene essentiality between different animal hosts?
most genes required in all three animals, but smaller substets specific to host species
salmonella uses some different genese in different envirnoments
- What are the aims and major findings of article 1?
goal: identify the Salmonella genes that contribute to long term systemic infections
118 genes were identified
30% HGT regions (pathogenicity islands, prophages, virulence plasmids)
genes that affectes membrane structure
Competitive Index: some of the genes (secreted SPI2 effectors, SPI4 locus (salmonella pathogenicity islands)) that we thought were needed were downregulated and instead a type 3 secretion system was upregulated -> challenges assumptions about what we thought we knew
- What are the aims and major findings of article 2
study genetic basis of intestinal persistence and zoonotic transmission
pools of random insertion mutations of Typhimurium in chickens, pigs, cattle with TraDIS
2715 genes with phenotypes
allowed them to assign geno- and phenotype of oer 90% of mutants, thereby annotate genomes with functional annotation
- How does Microarray-based negative selection screen work?
input
pooled samples (liver spleen)
all samples were hybridized against a common reference in which gDNA from a separate overnight cultre of the library was used as the template for the in vitro transcription reactions
Negative significant genes are genes whose mutants are absent in the tisse sample but present in the input
- How does TraDIS work?
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