Background and genetic techniques

Question 1

Botulinum toxin

Answer 1

Blocks neurotransmitter release in neuromuscular junction
Causes flaccid paralysis

Muscles relax –> Eventually host suffocates

LD50 = 1ng/kg of body weight

Question 2

Tetanus toxin

Answer 2

Produces by Clostridium tetani
Prevents reuptake of neurotransmitter
- continuous firing of neuromuscular junction
- every muscle in body contracts at the same time

LD50 is 2.5 ng/kg od body weight

Question 3

Reverse genetics

Answer 3

Seeks to assign function to a gene/sequence

Start with hypothesis
Uses directed mutagenesis
Hypothesis directs experimental analysis

Question 4

How to make a knockout?

Answer 4

Insertion
- disrupt open reading frame

Deletion
- delete genes so coding sequence in entirely absent

Question 5

Techniques for making knockout

Answer 5

Lambda red
Group II interferons
Homologous recombination, etc

All involve making a version of the gene you want in the lab and convincing bacteria to swap their version for new one

Question 6

How to complement?

Answer 6

1. Put gene back in on a plasmid
2. Insert gene somewhere else in genome

Insertion is better as you put a single copy of the gene back in so will get expressed similarly to how it would be naturally
- plasmids have higher copy number

Question 7

Forward genetics

Answer 7

Start with phenotype but need to work out what genes are responsible
Uses random mutagenesis

Experimental approach designed to screen for a phenotype

Requires thousandssssssssssss of mutants

Question 8

Random Mutagenesis

Answer 8

Largely relies on transposons
- random insertion into genome
- screen library of mutants using TraDIS

Question 9

TraDIS

Answer 9

Transposon directed insertion site sequencing
Transposon with antibiotic resistance cassette randomly jumps into bacterial genome
- large pool of bacteria
- each cell has different transposon insertion site

Break up bacterial cells; share DNA
Sequence transposon out of genome

Map different reads to bacterial reference genome

Essential genes will have no transposon hits

Question 10

Studying composition of microbiota

Answer 10

Sequencing
Illumina or Nanopore
16S rRNA sequencing
Metagenomics

Question 11

Kitome

Answer 11

Bacteria are present on kit used for studying microbiome
- led to false claims of microbiomes within different areas i.e. placenta

Question 12

Factors affecting composition of microbiome

Answer 12

Age
Diet
Gender
Invasive species
Antibiotics

Often links between them

Question 13

Role of microbiome for humans

Answer 13

Train immune system

Colonisation protection

Metabolic function

Question 14

Consequences of microbiome disruption

Answer 14

Obesity
Type II diabetes

Chronic inflammation

Loss of colonisation resistance
- infectious disease