L14: Culture Independant Microbiology Flashcards
1
Q
What are the benefits of environmental microbiology
A
- in lab
- bypass limitations of culture based by extracting nucleic acids directly
- manipulate + chartacterise individual DNA + RNA molecules from complex microbial communities
- helps moniter populations
- helps with biodegredative pathways/ biogeochemcal cycling
2
Q
What are the 4 types of molecular biology techniques
A
- nucleic acid analysis
- liquid profile analyeses
- fluorescent in situ hybridiation
- linking phylogentics to functions
3
Q
What are the steps in nuclec acid analsys
A
- extraction of total community nucleic acids from environmental samples
- PCR dependant methodologies
- methodologies to characterise PCR amplification products (cloning + sequencing of 16S rRNA fragments)
4
Q
What makes good nucleic acid analyses
A
- reliability
*yield - purity
- shearing degree
5
Q
Compare extraction of DNA, proteins, enzyme, mRNA
A
- can extract DNA cus stable + proteins enzymes, mRNA is harder and rapidly degraded
6
Q
Step 1 of nucleic acid analyses (aim, main component, main success, success depends on)
A
aim: high quality/quantitiy extract to provide snapshop of indigenous microbial community
main component: successful extraction + purification of a good amount of NA
main success: soil DNA isolation good in reliability, yield, purity, shearing degree
what does success depend on: source of sample, co-extracted interfering substances like heavy metals, pollutants)
7
Q
What are 2 general methods in nucleic acid extraction
A
a. direct lysis of microbial cells by chemical treatments, freeze-thaw, sonication or beatng => DNA shearing
b. extraction of bacterial cerls from the samples then cell lysis
8
Q
What is often extracted among soil, what happens
A
- humic + fulvic acids are co-extracted from soil with NA
- must be removed cus it interferes with next moleculare reaction
- DNA polymerae amplification
- DNA DNA hydrbirdation
- DNA labelling
- Restriction nuclease digestion
9
Q
What are the problems in nucleic acid extraction, + fix
A
- physical, chemical, biological step in prep is source of bias
- was there suffucient/preferential disruption of microbial cells
- rigorious conidtion for gram+ lyse could lead to excessive shearing of gram= cells
- reported diversity of sample becomes bias
- creates artifcants + chimeric PCR products
- fix: checking soil via argose gel electro indicate quality and extent of sheeing
10
Q
How can nucleic acids be purified
A
- chromatography
- silicia gel columnPVPP spin filter
- magnetic bands
11
Q
How does quantification take place + problems
A
- quality + quantity by argose gel electro
- spectrometry ⇒ nanodrop
- fluoremeter ⇒ qubit
- problem: spectrometry is inaccurte in comparison
12
Q
Why is it PCR dependant called that, and what is the main objective
A
- name: cus reliatn on PCR amplification
- obj: amplify target gene sequences from community so they can be characterised
13
Q
What are the steps in the PCR procedure
A
- denaturation: dsDNA seperarted to ss at high temps (denatures)
- annealing: two oligonucleotide primers anneal at lower temps to complementary regions that flank the target sequence of ssDNA
- Extension: heat stable DNA polymerase sytheises new strand be extending the primer using complementary strand as a template ⇒ makes duplicate copy of the target sequence
- repeated 20-30 times = exponentioal amplifcation of target
14
Q
What is amplified, with examples
A
- functional genes of interest
- contaminated soil: catabolic genotypes to see biodegradative genotypes to see functional microbial populations to see the appropiate strategies
- phylogentic/taxonomic genes of interest
- two types or rRNA in ribosomes(pro: 16s, 23s, 5s // euk: 18s,28s,5.8s)
15
Q
Describe ribosomal components
A
- 30S: small subunit
- 16s + 21 proteins
- 50s: large subunit
- 23S + 5S + 30 proteins
16
Q
What makes rRNA good chronometer + phylogenetic markers
A
- 16s highly conserevd and is essential, low mutation
- also has signature sequence motifs that is hypervariable and differentiates phylogentic distances
17
Q
List other phylogenetic markers
A
- chaperonin 60
- rpoB
- gyrB coding for topoisomerase II
18
Q
How are PCR primers selected
A
- based on superkingdom, family, genus, subspecies
- cover many conservaed + variable regions
19
Q
What is the routine procedure in 16S rRNA sequencing
A
- amplification of gene encoding 16S rRNA = PCR
- sequencing amplified genes = illuminia miseq
- analyzing of sequence in ref to other sequenences ⇒ Bioindormatics
- Identify microorganisms bases on 16S rRNA sequences, determine community composition
- Based on identities, extrapolate function / metabolisms of the microbial community inhabiting / detected in a given sample
- Use bioinformatics / ecological statistical methods to determine diversity, compare similarity between samples
20
Q
What happens in 16S sequence comparison
A
- when samples are sequenced, you submit to blast and fasta for comparison
- they approximate Smith Waterman but needs a lot of computing power
- want similar hits, top e-value otherwise study conserved regions, variable regions, chimeras
21
Q
FASTA
A
fast approximation
22
Q
How does tree analysis work
A
- distances are relative, distance is relative to all others in the set
- nodes spin to analyse relationships
- bootstrap to determine phylogenetic accuracy by resampling dataset + % of times group occurs
23
Q
What is the bootstrap method of tree analysis
A
dataset resampled and % of times group occurs is calculated
24
Q
What is the caveat of rRNA
A
rrn operon: 16S, 23S, 5S, internal spacer + 1 tRNA
25
How many rRNA operons can baccterium posses
- rrn copy numbers:
- bacterial genomes have max 15 operons
- 7 is most common
- 40% organisms have 1-2
- divergence among operons is small, <1% nucleotide difference
- very few (5) with higher level nucleotide divergence is detected
- in themophilic bacteria meaning HGT probably happended in this type
26
What is the role of rRNA in relationships
Genome sequences currently confirm that the rRNAs provide a relatively solid framework for the estimation of phylogenetic relationships
27
Go deep into step 3 of nucleic acids analyses
Step 3 = methodologies to characterise PCR amplification products: cloning and sequencing of 16S rRNA fragments
28
What are the steps in cloning + sequencing 16S rRNA fragments
- 16S rRNa is PCR amplified then cloned to PCR fragment vector to make clone library
- unique clones are screened by ARDRA which is a RFLP
- the 16S rRNA fragments are sequenced and origin/identification determented by BLAST or FASTA
29
What is the general approach for cloning and sequencing 16s fragments
- compare SSU rRNA genes (16 for pro, 18 for euks)
- SSU rRNA gene similarity isued to define OTU of working species and diveristy is measured by rarefaction curve
30
What is OTU and how is species diversity measures
- operational taxonomic units to measure gene similarity
- rarefraction curve for species diversity
- plotting # of OTUs as function of increasing sample size
31
List the phlyogentic composition of bacteria 16S rRNA clone library
- high GC gram positive
- delta-proetbacteria
- alpha proteobacteria
- CFB
32
What are examples of new technologies and what do they help us do
- 454 pyroseqeuncing, illuminia, oxfoed nanpore
- generates larger 16s rRNa database
33
What is the problem with PCR-Dependant Techniques
1. very dependent of PCR primers and can be too specific
2. only looks at what we already know
3. PCR amplifies errors
4. hard to quantify genotypes for environmental samples BUT qPCR is making it better
5. only amplifies limited regions ⇒ limits the amount of useful sequencing information
34
What are other molecular techniques in env microbio
- nucleic acid analyses
- lipid profile analyses
- FISH
35
What are examples of lipid profile analyses
- FAME analysis by GC (gas chromatrography)
- based on extraction of membrane lipid components
- then you seperate, identify and quantificaton of membrane fatty asids
- good for new species, using fatty acids as biomarkers
36
What is FISH
- fluorescent in sity hybridisation
- microscopic tech using biofilrms
- put sample on slides and cells are fixed
- specific cells are hydrbised with specific fluorescently labeled probe targeting 16S
- singal detected + quantified by fluroecent microsopy/CSLM
- determine composition + spatial distribution in situ
- enchamces signal CARD-FISH
37
What methods are used to linking phylogenetics to function
1. stable isotope probing (SIP) technique
2. MAR-microautoradiography-FISH
38
What is SIP tehcnique
- identifies specific groups resposible for in situ biogeochemical tranformation process
- identify active members in communities (methanogens…) by linking metabolic activity to diveristy using isotopes
- microorganisms metabolisim a stable isotope incorportate it into DNA
39
What is MAR Fish
- use radioisotopes to measure microbial activity
- follows in vivo uptake of radiolabeled substraces with speicifc organism idenitified by fish
- linking specific genes and function to specific organisms
- combines phylogenty activity of cells
