Lecture 4 - Microbiome Flashcards
What is a Microbiome?
(3 Points)
- Catalogue of Microorganism species are present
- Catalogue of what genes are present
- Knowledge of how the microbiome changes in response to stimuli
How can microbiomes be divided?
(2 Points)
- Physiological - e.g., Oral, Gut, Skin
- Ecological - e.g., City, Environment
What are the 3 Main Stages of Microbiome Projects?
- Who is there? - Analysis of Cells (FACS), DNA/RNA (Sequencing)
- What are they doing? - function of genes present in Microbiome
- How do they do it? - Mechanism of Action
How are Species/Gene Families of Organisms present within a microbiome determined?
(4 Points)
- DNA Sequencing - to find specific genes (e.g., Antibiotic Resistance)
- Metagenomics DNA Sequencing - all sample DNA
- Transcriptomics - mRNA Sequencing (Active Genes)
- Cell Sorting (Dead or Alive)
How can DNA Sequencing be utilised for Taxonomy?
By comparing the sequence of a single gene that is highly conserved (e.g., 16S rRNA) across multiple organisms, you can obtain a good measure of how related two organisms are
Describe the workflow involved in metagenomics DNA sequencing
(4 Points)
- Fragment sample nucleic acids to length suitable for PCR and Sequencing (<1kb)
- Convert nucleic acids to dsDNA, often with poly-A overhangs generated in RT-PCR
- Attach Oligonucleotide adaptors to poly-A tails of DNA fragments (Allow identification of origin)
- Final Library product is ready for sequencing by Illumina machines or nanopores
Describe the Process of Fluorescent Activated Cell Sorting (FACS)
(3 Points)
- Cells are mixed with fluorescent stain e.g., DAPI (binds to nucleic acids)
- FACS machine shines laser to detect distribution of DAPI
* If cell is alive, their nucleoid will be intact and therefore image will be compact
State the Human Microbiome Hypothesis
Microbiome is an entire organ of the human body, with its activity and fluctations influencing human health
Describe the General composition of the Human gut Microbiome
(2 Points)
Consists of:
* Primarily Bacterial species (e.g., E. coli, Roseburia)
* Few species of Archaea (e.g., Methanobrevibacter)
How does the Firmicutes : Bacteroidetes ratio impact patient health
High Firmicutes : Bacteroidetes ratio and low diversity results in obesity and a high calorie diet
How was the “Core Human Gut Microbiome” Hypothesis Disproven?
(2 Points)
- From 154 gut samples, no single bacterial phylotype was detectable at an abundant frequency
- However there was constancy in the conserved orthologue genes (COGs) irrespective of species present
How does Microbiota Genetics influence our metabolism/health?
(2 Points)
- Diet introduces specific food chemicals into the gut microbiome, which selects for microbes with genes capable of utilising the specific chemicals
- Microbial metabolism generates new molecules, which have physiological consequences and can impact our health
(i) How is Dietary fibre converted into Butyrate?
(ii) What are its benefits?
(i) Fibre consists of Xylan/Glycan Carbohydrates, which can be metabolised by some bacterial species that possess Polysaccharide Utilising Loci (PULs)
(ii) Butyrate is released from the bacteria and taken up by the gut, where it has been suggested to generate positive anti-inflammatory responses (triggers inflammation)
(i) What are Starch Utilisation System (sus) genes?
(ii) Give two examples and their functions
(i) Bacteroidete genes present in PULs which allow them to metabolise xylans/glycans
(ii) Examples:
* SusC - mediates transport of starch oligosaccharides from surface of OM into periplasm
* SusD - mediates starch binding before transport into periplasm
What are the functions of sus gene products?
- Bind polysaccharides
- Hydrolyse polysaccharides/oligosaccharides
- Import oligosaccharides/monosaccharides
