techniques

Question 1

biochemial approach

Answer 1

• used to find the virulence factors
• mostly looking for toxins produced by your pathogen
• technique: the culture supernatant is fractionated. then it is injected into an animal model to see if it will cause damage to the model.
• can purify and inactivate the virulence factor to create a vaccine
• (goal is not to find a gene)

Question 2

cloning

Answer 2

goal: make the nonpathogenic bacteria a pathogenic bacteria (looking for one virulence factor)
- make a library of plasmids that all have a different piece of the pathogenic strain genome, insert these into the nonpathogenic strain, and then screen the bacteria for the phenotype you are looking for, once you find it, take the plasmid out and sequence the DNA fragments to find the gene
- limitations: can only insert less than 30kb on a plasmid

Question 3

chemical or UV mutagenesis

Answer 3

• creates a library of mutants (via point mutation). then you screen them for the ability to cause disease. identify mutants that lost the traits.
• take the mutant that (cannot grow inside cells) and then sequence it to see what is missing or different etc.

Question 4

transposon mutagenesis

Answer 4

• create a library of transposons

- identify the gene that was interrupted by a transposon by doing whole genome sequencing

Question 5

transposons

Answer 5

-jumps around the chromosomes. when it is inserted inside a gene, the gene will not be expressed.

Question 6

goals of genome sequencing

Answer 6

• find PAIs
• compare genomes
• identify genes
• find homologue of virulence factor

Question 7

RNA -SEQ

Answer 7

identify genes EXPRESSED during disease/infection

• that piece of RNA comes from that gene etc
• more gene products = greater expression of that gene