How we study the Microbiome Pt 1
Traditionally we focused on visualisation techniques – growing in the laboratory, microscopy etc. Now we use DNA and new sequencing technology. We can see everything without requirement for cultivation in the lab.
The Dutch Antonie Philips van Leeuwenhoek was the first microbiologist and invented microscopes to look at teeth. The Englishman Robert Hook was also an early pioneer. Microscopes evolved and staining techniques were developed. German Robert Koch, a pioneering microbiologist, was the first to grow bacteria on solid media. In the 1880’s he made it possible to separate different kind of bacteria and proved that microbes cause disease.
These developments allowed us to:
- Isolate bacteria
- Culture the pathogen
- Infect healthy host
- Re-isolate the bacteria
What is the culture of microbes?
This is the growth of microbes in a media that will support them. Single cells develop into a colony. Some mediums only support one kind of bacteria while others support many. Dyes are used to separate the bacteria.
A big problem is that not many bacteria could be cultured, also those that grow well in culture might not be the most important or abundant in that particular habitat. E-coli is the most readily cultured but is actually not that abundant in the gut. The ‘Great Plate Count Anomaly’ discovered by Staley and Konopka describes this difference between the microbes we can successfully grow in a culture and those we can observe. Only 0.1% – 1% of microscopic bacteria are cultured.
The use of DNA
Carl Woese was the first to compare DNA sequences to determine the relationship of microbes between each other, using ribosomal RNA genes.
The amount of sequence difference between organisms corresponds to the amount of evolution between organisms. ‘Woese Maps’ made it possible to determine what kind of microbes live in different environments. Culture was not necessary to isolate DNA.
- Environmental sample processed to extract DNA
- Ribosomal RNA gene isolated and sequences determined
- Sequences compared to collections of known sequences to identify microbes
Why was this such an important development?
- We can investigate whole microbial communities
- This approach is used to study many environments from hot springs to the human microbiome
- Many organisms not detected by culture dominate their respective environment.
- We are able to significantly expand our knowledge of microbes from the Bacteria, Archaea and Eukarya Phyla (groups)
In the 1980s only 12 bacteria phyla were known and all had cultured representation. In 20 years we have expanded to 100 Phyla, 2/3rds of which have no cultured examples and are only detected by DNA sequencing.
Basics of high-throughput DNA sequencing
Morphology is the branch of biology dealing with form and structure
There are two problems
- It is difficult to distinguish microbes using morphology
- The majority of microbes are hard to grow (especially gut microbes)
DNA solves both these problems
What is DNA?
DNA is a molecule found in humans and all living organisms which stores information about our bodies and how they work in a code.
The code is a sequence of 4 chemical bases/nucleotides: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). These have a phosphate backbone in 2 long strands.
They are paired as either A and T or C and G
Genome: this is all the DNA in any one organism and is the blueprint that cells use. Within the genome, DNA is organised into genes.
Genes are regions of the genome that correspond to a unit of inheritance i.e. eye colour/hair colour/blood type. A gene is a single part of the entire blueprint of a cell. To pass on information, genes are transcribed into messages and passed on by proteins.Some genes are the same in all organisms, others vary and result in differences. In microbes genes may control which compounds can be used as food
Proteins do most of the work in the body and are machines that blueprint and provide instructions for cells
The areas of a gene that are variable drive the evolution of a species
Method of analysis by DNA
- Target gene present in all microbial community members
- Looking at conserved regions: finds all microbes
- Looking at variable regions: distinguishes different types of microbes
- The 16S rRNA gene is found in all bacteria and archaea. Humans don’t have this gene. It is involved in the creation of proteins from DNA and therefore it is very important
- 16S rRNA is 1500 nucleotides long with 6 regions (some conserved and some variable)
- The V4 region helps to tell them apart
Using 16S to distinguish microbes
- Extract DNA
- Make copies of V4 region
- Sequence 16S DNA
- Use sequences to distinguish microbes from each other
DNA extraction is the physical and chemical methods that break cell membranes and let DNA out. Material (in the case of gut microbes this is faecal material) is ground up and shaken using detergent to poke holes in the cell membranes. Other proteins and contaminants are removed. The DNA is then cleaned for analysis.
Copies of the region are then made.
We are not limited to one type of DNA. When we extract DNA from the faecal sample, we get DNA not just from microbes but also from things such as plants people ate or other cells. We make copies of the microbial DNA and then discard the rest.
PCR – Polymerase Chain Reaction (mimics how DNA is copied in nature)
To copy itself, DNA has to unwind. The enzyme that unzips DNA is called helicase. Polymerase is the enzyme that adds complimentary base pairs. There are two copies of the DNA – each made up of half old and half new.
Polymerase needs certain requirements to work
- Single stranded DNA to serve as a template
- A starting place where DNA goes from double-stranded to unwound and single-stranded
- Free nucleotides – AGCTs
PCR is the lab synthesis of DNA from a template. It needs similar requirements to nature. It needs a starting point (using oligonucleotide primers). These are small pieces of DNA bought in from a mail order company. These show the polymerase where to start and stop copying. This is used to target the different areas of the gene regions. Primers are located before and after region and the polymerase makes copies of sequences in between the primers.
- Pieces are mixed in the tube
- Put through hot and cold temperature cycles
- Heated to make the DNA unwind
- Coding lets primers attach to target region
- Goes through many cycles and copies increase exponentially.
Next the samples are purified and sequenced (copies are made and keep track of every nucleotide added).
Dye is used for the nucleotides so that only one is added at a time, each a different colour. The colours are tracked in order which gives the DNA sequence using high-throughput sequencers. This can be done by hand but slow and inaccurate, so now it is carried out by robots.