Forward and Reverse Genetic Approaches to understanding Bacterial Pathogenesis Flashcards
(24 cards)
Reverse Genetic Technique (OUTLINE):
Seeks to assign function to particular gene:
Start with hypothesis -> directed mutagenesis (either knockout or subtle mutations) -> Hypothesis directs experimental analysis (phenotype tested)
- Process usually backed up by another data source e.g. biochemical assay
Forward Genetic Technique (OUTLINE):
Seeks to identify genetic basis of a phenotype:
No prior knowledge (phenotype / symptom) -> random mutagenesis -> experimental approach designed to screen for phenotype -> Investigate screened mutants for common genes.
What are the two main mutation options to form a knockout by mutagenesis?
Insertion (disrupts reading frame, can disturb regulons or multi subunit proteins) and Deletion (coding sequence removed). The choice of which is used is usually species dependent.
Why do most mutagenesis methods co-opt natural systems?
Nature is more efficient at developing molecular biology tools.
Examples of mutagenesis techniques:
Lambda Red, Group II Introns (TargeTron), Homologous recombination, Phage transduction, CRISPR, Transposons,
What are the two main complement options?
Plasmid (insertion of plasmid into the cell) or insertion at distal locus (insertion of single gene into plasmid)
What is required for complementation by plasmid?
Selectable markers, origins of replication, transformation/ conjugation/ promoters, etc.
What is required for complementation by insertion?
A suitable insertion site, homologous recombination machinery, an unstable/conditional plasmid, counter selection, etc.
Which of the complementation methods is preffered?
Insertion mutagenesis, as expression is more often similar to the wild type. Plasmid mutagenesis may leads to overexpression of proteins (due to the possible incorporation of multiple plasmids simultaneously)
How many transposon mutants can be screened in TraDIS/TnSeq?
50,000+ in a single pool.
What is TraDIS? (Name)
Transposon Directed Insertion site Sequencing
What are the steps of Tradis?
Generation of a Transposon Library -> Generation of Transposon Mutant Pool (mutagenesis) -> DNA extraction from surviving colonies -> Fragments are sequenced using Illumina (next gen seq) -> Insertions mapped to reference genome
How do you identify essential genes from a TraDIS insertion map?
Gaps in insertion presence, as no knockouts survived with those genes knocked out
How can Tradis be used to identify conditionally essential genes?
When culturing your transposon mutant pool, you culture them and expose them to a selection pressure. After sequencing the fragments the insertion map can be compared to that of mutants not in the stress environment. Additional gaps appearing in the stressed sample represent genes required for growth in the stress condition.
Application of TraDIS in C. difficile:
-> Spores purified using a HistoDenz gradient -> then germinated in broth with taurocholate -> Mutants incapable of sporulation would be absent from the purified spore population -> causing additional insertion gaps in genes required for sporulation (when compared to normal growth transposon insertion sites)
What strain was used in TraDIS investigation of C. difficile?
The highly virulent epidemic strain R20291 (ribotype 027)
How many genes were identified as essential in C. diff using TraDIS?
404 essential genes and 798 sporulation genes
Why should sporulation genes be considered essential for C. diff?
C. diff is anaerobic, and-so its’ primary transmission form is as a spore, otherwise it’d die in the many aerobic environments it’d cross during transmission.
Examples of Next-Gen Sequencing techniques:
Illumina (short reads)
Nanopore and PacBio (long read)
These methods can be used to sequence the DNA at a high throughput, by sequencing in parallel.
Steps of identifying the species in the microbiota:
- Amplify genes encoding 16S ribosome RNA -> extract these genes and sequence the fragments -> Identify variants of 16S rRNA (cross-referencing known databases)
Why is 16S ribosomal RNA used to identify species in the microbiota?
16S rRNA is highly conserved subunit of RNA, however drift between species allows for the identification of most species in a mixed sample.
How can the relative populations (proportion) of bacterial species in the microbiota be predicted?
By sequencing the 16S rRNA sequences in the mixed sample, and calculating the frequency of each 16S variant within the population.
How can characteristics of the microbiota be determined? Without being able to be cultured or determined by 16S rRNA freq.
Shot gun sequencing of all DNA in the sample -> genome sequences reassembled -> some gene regions will be identified (e.g. resistance to antibiotics by members within the population)
