Practical papers

Question 1

To discover genes involved in virulence

Answer 1

Genomic library
Transposon mutagenesis
High-through put screens
Functional genomics
Bioinformatics in silico experiments.

Question 2

Exploring importance of virulence genes.

Answer 2

Molecular epidemiology
Mutational analysis
Try as a vaccine
Structure/function studies
Cell biology studies

Question 3

Using the genomic library to screen for virulence

Answer 3

Introduce sections of genome into plasmids, express in bacteria and screen for virulence.

Question 4

Transposon mutagenesis: transposons.

Answer 4

. A transposon is a mobile genetic element that can insert into DNA. They are flanked by direct repeat sequences, contain two insertion sequences and carry a transposase and possibly other genes too.

Question 5

Advantages of transposon mutagenesis over normal mutagenesis.

Answer 5

 Only one insertion
 Insertions are random
 Insertions are stable
 Mutants can be selected by inclusion of an antibiotic gene.
 Easily identifiable transposon –> easy to identify mutated gene.

Question 6

Transposon mutagenesis technique using antibiotic resistance.

Answer 6

o Have bacteria carrying plasmid with resistance to one antibiotic e.g. chloramphenicol on a temperature sensitive plasmid. Recipient cells have chromosomal resistance to another e.g. streptomycin.
o Mating between the two.
o Now have a selection: donor cells which are vulnerable to streptomycin and to high temperature. Donor cells where the transposon hasn’t integrated which are temperature sensitive, and donor cells where it has integrated which are not (also resistant to streptomycin, chloramphenicol).

Question 7

Transposon mutagenesis technique using reporter genes.

Answer 7

Encode a clearly screenable phenotype.
o Insert transposon with promoter-less reporter gene e.g. promoter-less lacZY. Will remain unexpressed unless inserted into an active gene.
o Insert transposon lacking nuclear export signals: will only be exported if in gene for nuclear export. E.g. truncated PhoA.

Question 8

Specialised transposons for high-through put screening.

Answer 8

o Each signature tage as a unique central sequence of 40 bp, flanked by invariable arms of 20 bp, which allow amplification by PCR. You label each tag, and then ligate one each to a transposon. These tagged transposons are used to mutagenise bacteria, which are pooled and then used to infect mice.
o Genomic DNA is isolated from the input pool for comparison.
o The output pool is compared to the input pool; DNA that hybridises to the input but not the output pool is important in virulence.

Question 9

Structure/function studies.

Answer 9

 Biochemical assays
 Protein-protein interactions
 Protein crystal structures

Question 10

Cell biology studies

Answer 10

 Tissue culture, microscopy to determine cellular localisation.

Question 11

Functional genomics

Answer 11

o Use next generation sequencing technologies.
o Compare genomes of pathogens with closely related non-pathogens, or with other pathogens.
o Use sequence diversity (SNPs, insertions/deletions) to reveal geographic diversity and to follow transmission.

Question 12

Transcriptomics

Answer 12

Studying pathogen gene expression
o Expression in host/environment, at different stages of infection, in different conditions etc etc.
o Microarrays using up to a million individual DNA probes attached to a glass surface, so up to 200 fold coverage of a single slide. Quantitative method to detect individual genes or transcripts, working on the basis of hybridisation.
o RNA sequencing is a more recent, more direct and quicker method. Requires you to isolate the RNA, reverse transcribe into cDNA and sequence.

Question 13

Bioinformatics can…

Answer 13

• Suggested function or activity
• Predict protein structure
• Suggest preferred cellular location of protein
• Indicate evolutionary origin.
Bioinformatics is based on sequence comparisons.

Question 14

Bioinformatics tools

Answer 14

BLAST (and variants)
Multiple alignment by TCoffee
ProSite database.

Question 15

BLAST

Answer 15

o Basic local alignment search tool. Similarity search.

Question 16

BLAST variants.

Answer 16

 BLASTP – protein query against protein database.
 BLASTN – DNA/RNA query against GenBank (DNA database)
 BLASTX – six frame DNA query against protein database.
 TBLASTN – protein query against 6-frame translation of GenBank database.

Question 17

Degrees of similarity in BLAST results.

Answer 17

 >25% = likely to be related
 15-25% = possibly related


Question 18

Multiple alignment by TCoffee.

Answer 18

o Identifies conserved patterns or motifs in protein primary sequences.

Question 19

ProSite database

Answer 19

o Identify protein motifs or domains linked to known functions (e.g. ATP binding etc). Especially helpful if known to be related to a proteins of certain function by BLAST.

Question 20

Pull-down assay.

Answer 20

• Fuse affinity tag to bacterial virulence factor and bind to beads.
• Mix host cell lysate with bait.
• Wash off all unbound host proteins.
• Cleave bacterial protein – host protein complex from tag using highly specific protease and analyse eluted proteins by mass spec.

Question 21

Fluorescence microscopy of live cells

Answer 21

o Tag genes of interest with GFP from jellyfish or RFP from coral, or engineered variants of natural fluorescent proteins.

Question 22

Fluorescence microscopy of dead cells.

Answer 22

o Immunofluorescence microscopy carried out on chemically fixed and permeabilised host cells. Cells infected with pathogenic bacteria or transfected with gene of interest.
o Treated with antibody specific to that virulence factor which has been linked to a fluorescent dye.
o Fluorescence is observed under UV light.

Question 23

Multiplicity of infection

Answer 23

The number of virions that are added per cell during infection. This is m in the Poisson distribution.

Question 24

Influenza haemagglutination assay

Answer 24

HA binds to sialic acid receptors on cells. The virus will also bind to erythrocytes (red blood cells), causing the formation of a lattice. This property is called hemagglutination, and is the basis of a rapid assay to determine levels of influenza virus present in a sample.

Question 25

The plaque assay

Answer 25

Only plates between 10 and 100 plaques are counted. Duplication to enhance accuracy.

Question 26

Endpoint dilution assay

Answer 26

Serial dilutions of a virus stock are prepared and inoculated onto replicate cell cultures, often in multi-well formats (e.g. 96 well plastic plates). The number of cell cultures that are infected is then determined for each virus dilution, usually by looking for cytopathic effect.
The dilution at which half of the cell cultures are infected can give you the ID50 per millilitre.

Question 27

The western blot

Answer 27

The sample is solubilized with detergent, and the proteins are then separated by electrophoresis in a polyacrylamide gel. After electrophoresis, the gel is placed next to a thin, synthetic membrane that has a strong affinity for proteins.
Proteins in the gel are transferred to the membrane by capillary action.
The membrane is incubated with an antibody to a specific protein.
The antibody may be coupled to an enzyme which can then be used to detect the antibody on the membrane.

Question 28

Immunostaining.

Answer 28

An antibody that recognizes a viral antigen is coupled directly to an indicator in direct immunostaining. Indirect immunostaining is a more sensitive method because a second antibody is coupled to the indicator.
Cells are fixed.
Used to determine subcellular location of cells.

Question 29

Viral recombination

Answer 29

Recombination analysis can be used to identify origins of recombinant sequences.