4: Which genetic technique can you use to identify virulence factors? Flashcards
What is a virulence factor?
Any (gene) product that is involved in the survival and persistence in the host.
What is a pathogenicity factor?
Any (gene) product that is involved in damaging host tissue.
Which methods are there to find virulence?
Educated guess (directed mutagenesis), genetic screen (random mutagenesis) or comparative genomics.
How do you make directed mutations?
By reverse genetics, with a single-crossover event or double-crossover event. In a single-crossover event there is reversed mutation possible, and in a double-crossover event it is not possible so that method is prevered.
You can also use CRISPR-Cas.
What is the disadvantage of the educated guess method?
It is an high amount of work, and ‘what you know is what you get’. It is a good approach for viruses.
How do you make random mutations?
Bij forward genetics, with chemial mutagenesis, UV irradiation or transposon mutagenesis.
What is a transposon?
A transposable sequence that can change it’s position within a genome by recombination, sometimes creating or reversing mutations and altering the cell’s genetic identity and genome size.
What are the advantages of transposon mutagenesis over the other genetic screen mutations?
gene disruption, every clone contains a mutation, every clone contains a single mutations and the mutations are easy to identify.
What is a double-filter immunoblot screen?
Double filter immunoblot screen shows the amount of proteins secreted by bacteria.
What is TraDIS?
Transposon directed insertion-site sequencing, you select 100.000 different transposon mutants so you have a full genome coverage of transposons, and then you put them in a mouse and can determine all infection-deficient mutants in the mouse.
What is the advantage of traDIS?
It is genome wide, you know the direct location of the transposon and it is easy to quantify.
Why do you have to be carefull of bottlenecks in transposon mutagenesis?
Bottlenecks can cause a mutant to be lost by chance and that doesn’t mean it is a virulence factor. So you should use enough bacteria to not have a small bottleneck.
What is the wild-type helper effect?
Most cells behave like wild-type cells, and in mixtures the toxins that the wild type cells produce can help the mutants.
Why are mini transposons preferred?
Because normal transposons can jump multiple times and mini-transposons can’t, they lose their gene after one jump.
