Lc2: Microbial techniques

Question 1

culture

Answer 1

• Culturable fraction = +- 30%
• So we could not study the complete intestinal microbiota
• Culture-independent techniques required
• Issues:
  o Slow microbial growth
  o Low abundance of microbe
  o Inhibition by other microbes in culture
  o Co-cultivate strains
  o Growth factors are absent/unknown

Question 2

DNA approaches

Answer 2

• gene amplicon –> identification
• shotgun metagenomics –> what can they do and who they are (whole genome) (used for uncultured microbes)

Question 3

RNA approaches

Answer 3

metatranscriptomics –> what are the bacteria doing?

Question 4

protein approaches

Answer 4

metaproteomics –> what are the bacteria doing?

Question 5

metabolite

Answer 5

metabolomics –> what are they doing?

Question 6

next-gen sequencing

Answer 6

1. DNA is fragmented and adaptors are added which can form bridges by connecting to primers on a flow cell (glass plate)
2. PCR extension occurs of the fragments
3. The bridges dissociate and a new PCR cycle can start
4. Clusters of the original fragments can now be seen
5. Now fluorescent nucleotides will be added one by one to do the sequencing

Question 7

signature rRNA

Answer 7

• The 16S rRNA gene can be used to identify any microbe
• Present in all bacteria & archaea
• Highly variable regions
• Interspersed with conserved regions