MBB 267 Week 5: Corrigan 1

Question 1

What are the advantages of using microbes for genetic testing?

Answer 1

Include:

a. Rapid Reproduction: large number produced in short periods of time
i. These populations are large enough to contain spontaneous mutants
b. Simple to maintain and cultaivat

Question 2

What are the advantages of using bacteria for genetic testing?

Answer 2

Bacteria are haploid – phenotype of mutation seen immediately

a. Relatively small genome (485 genes in Mycoplasma genitalium, ca. 4,000 genes in E. coli)
b. Genetic manipulation (e.g. gene knockouts) straightforward
i. We can make strains carrying desired combinations of mutations with relative ease

Question 3

What is the classical (forward) genetic approach for testing? and how does it work?

Answer 3

Forwards = biological function (phenotype) to gene
a. First we observe the mutation then we look for the gene sequence
b. Phenotypic screening for desired mutants
Biochemical/physiological characterisation of the mutants
Genetic analysis (genetic mapping - sequencing, complementation test) Gene isolation - Study gene product

Question 4

What are the advantages of the forward genetics testing approach?

Answer 4

Advantage: We already know the mutation = Emphasis is on the desired phenotype (e.g. loss of pathogenicity or growth)
Can find mutants with defects in essential genes (conditional lethal)

Question 5

What are the disadvantages of the forward genetics testing approach?

Answer 5

Disadvantage: slow; may be impossible to find all the genes in a species for a given phenotype

Question 6

How does reverse genetics work?

Answer 6

We make the gene then we observe the function

a. From gene to phenotype by altering the gene sequence or inhibiting its expression
b. This is currently Possible due to the advancement of molecular genetics

Question 7

What is the procedure of reverse genetics experiments?

Answer 7

we know the entire microbial genome sequences

a. Focus on one gene of particular interest e.g. has homology to a gene of known function in another microbe;
b. Ask what is the role of each gene…
i. Mutate gene (DNA) in vitro
ii. Substitute the mutated allele for the wild-type allele in genome
iii. Determine phenotype of resulting mutant strain

Question 8

How are phenotypes identified for different mutants/ conditions?

Answer 8

Use a BiOLOG phenotype array
20 x 96 well plates, all with a defined media that can be altered eg 96 different carbon sources
a. or 96 different sulphur sources
or some wells have an antibiotic in them
b. Then we compare the well from the WT bacteria and the mutant bacteria

Question 9

What are the uses of mutants?

Answer 9

MUTANTS HELP TO IDENTIFY GENES INVOLVED IN A PARTICULAR FUNCTION

a. PERMIT UNDERSTANDING OF METABOLIC REGULATION
- i. e.g. mutants of transcription factors that lead to upregulation of a gene
b. Can help us to find which gene block a certian intermediate in a pathway
c. Help us to undersand metabolic regulation
- i. Mutantions in transcritpoinal factors lead to up/downregulations of genes
d. IDENTIFYING THE SITE OF ACTION OF AN ANTIBIOTIC
e. Find Conditional lethal mutations
- i. When the mutation phenotype is seen under certain conditions
f. Can help us to clone genes

Question 10

What are the types of mutations?

Answer 10

Include

a. Point mutations: change in one base pair
i. Transitions; Base changes (eg. Purine to purine) same base pair
ii. Transversions; Base changes to other base pair
iii. Insertions of portion of chromosome
iv. Deletions of potion of chromosome
v. Inversion a portion of chromosome